NO310294B1 - New inhibitors of thrombin - Google Patents

Abstract

This invention relates to novel biologically active molecules which bind to and inhibit thrombin. Specifically, these molecules are characterized by a thrombin anion-binding exosite association moiety (ABEAM); a linker portion of at least 18 ANGSTROM in length; and a thrombin catalytic site-directed moiety (CSDM). This invention also relates to compositions, combinations and methods which employ these molecules for therapeutic, prophylactic and diagnostic purposes.

Description

This invention relates to an analogue in the method for the production of new biologically active molecules, which bind to and inhibit thrombin which molecules have stretched as stated in claim 1's preamble. In particular, these molecules are characterized by a thrombin-anion-binding outwardly directed site-associating unit (ABEAM); a linker portion of at least 18Å in length; and a thrombin-catalytic set-directed unit (CSDM). The method according to the invention is characterized by the features that emerge from the characterizing part of claim 1.

Background technology

Acute vascular diseases, such as myocardial infarction, stroke, pulmonary embolism, deep-seated thrombosis, peripheral arterial occlusion and other blood system thrombosis pose a very large health risk. Such diseases are caused either by partial or total occlusion of a blood vessel by a blood clot containing fibrin and platelets.

Current methods of treating and preventing thrombotic diseases involve therapeutics that act in one of two different ways. The first type of therapeutic inhibits thrombin activity or thrombin formation and thus prevents clot formation. These drugs also inhibit platelet activation and aggregation. The second category of therapeutics accelerates thrombolysis and dissolves the blood clot, thereby removing it from the blood vessel and unblocking blood flow [J.P. Cazenave et al., Agents Action, 15, Su<p>pl., pp. 24-49 (1984)].

Heparin, a compound of the former class, has been used extensively to treat conditions such as venous thromboembolism, where thrombin activity is responsible for the development or propagation of a thrombus. Although heparin is effective, it produces many unwanted side effects including bleeding and thrombocytopenia. This has led to a search for a more specific and smaller one

toxic anticoagulant.

Hirudin is a naturally occurring polypeptide produced by the leech Hirudo medicinalis. This compound, which is synthesized in the leech's salivary glands, is the most powerful natural coagulation inhibitor known. Hirudin prevents the blood from clotting by binding tightly to thrombin (Kd = 2 x 10~1:LM) in a 1:1 stoichiometric complex [S.R. Stone and J. Hofsteenge, "Kinetics of the Inhibition of Thrombin by Hirudin", Biochemistry, 25, pp. 4622-28

(1986)].

This in turn inhibits thrombin from catalyzing the conversion of fibrinogen to fibrin (coagulant), as well as inhibiting all other thrombin-mediated processes [J.W. Fenton, II, "Regulation of Thrombin Generation and Functions", Semin. Thromb. Hemost.. 14, pp. 234-40 (1988)].

The actual binding between hirudin and thrombin is a two-step process. First, hirudin binds to a "low" affinity site on the thrombin molecule (Kd = 1 x 10"<8>M) which is separated from the catalytic site. This binding involves structural participation from the C-terminus of hirudin with an "anion -binding outward site" (ABE) in thrombin [J.W. Fenton, II et al., "Thrombin Anion Binding Exosite Interactions with Heparin and Various Polyanions", Ann. New York Acad. Sei., 556, pp. 158-65 (1989 )]. After the low-affinity binding, the hirudin-thrombin complex undergoes a conformational change, and hirudin then binds to the "high" affinity site in thrombin [S. Kono et al., "Analysis of Secondary Structure of Hirudin and the Conformational Change Upon Interaction with Thrombin", Arch. Biochem. Biophys.. 267, pp. 158-66 (1988)]. This latter site corresponds to the active site in thrombin.

The isolation, purification and chemical composition of hirudin are known in the art. [P. Walsmann and F. Markwardt, "Biochemical and Pharmacological Aspects of the Thrombin Inhibitor Hirudin", Pharmazie, 36, pp. 653-60 (1981)]. More recently, the complete amino acid sequence of the polypeptide has been elucidated [J. Dodt et al., "The Complete Covalent Structure of Hirudin: Localization of the Disulfide Bonds", Biol. Chem. Hoppe-Seyler, 366, pp. 379-85 (1985); S.J.T. Mao et al., "Rapid Purification and Revised Amino Terminal Sequence of Hirudin: A Specific Thrombin Inhibitor of the Blood-Sucking Leech", Anal. Biochem, 161, pp. 514-18 (1987); and R.P. Harvey et al., "Cloning and Expression of a cDNA Coding for the Anti-Coagulant Hirudin from the Bloodsucking Leech", Hirudo medicinalis", Proe. Nati. Acad. Sei. USA, 83, pp. 1084-88 (1986)] .

At least ten different isomorphic forms of hirudin have been sequenced and found to have a slightly different amino acid sequence [D. Tripier, "Hirudin: A Family of Iso-Proteins. Isolation and Sequence Determination of New Hirudins", Folia Haematol., 115, pp. 30-35

(1988)] . All forms of hirudin consist of a single polypeptide protein chain containing 65 or 66 amino acids, where the amino terminus primarily consists of hydrophobic amino acids, and the carboxy terminus normally consists of polar amino acids. Specifically, all forms of hirudin are characterized by an N-terminal domain (residues 1-39) stabilized by three disulfide bridges in a 1-2-, 3-5- and 4-6- hemicystenyl pattern and a strongly acidic C-terminal segment (residues 40-65). Moreover, the C-terminal segment in hirudin is characterized by the presence of a tyrosine residue at amino acid position 63, which is sulfated.

In animal studies, hirudin, isolated from leeches, has shown good efficacy in preventing venous thrombosis, vascular

shunt occlusion and thrombin-induced disseminated intravascular coagulation. In addition, hirudin has low toxicity, low antigenicity and a very short excretion

see time from the bloodstream [F. Markwardt et al., "Pharmacological Studies on the Antithrombotic Action of Hirudin in Experimental Animals", Thromb. Haemost., 47, pp. 226-29

(1982)] .

In efforts to obtain a larger supply of hirudin, attempts have been made to produce the polypeptide by recombinant DNA techniques. The presence of an O-sulfated tyrosine residue of native hirudin and the inability of the microorganisms to carry out a similar protein modification made the prospect of recombinant production of biologically active hirudin highly speculative. However, the observation that desulfatohirudins are almost as active as their sulfated counterparts (US patent 4,654,302) led to the cloning and expression of hirudin in E. coli [EP applications 158,564, 168,342 and 171,024] and yeast [EP application 200,655 ]. Despite these advances, hirudin is still relatively expensive to produce, and it is not widely available commercially.

In recent times, efforts have been made to identify peptide fragments in natural hirudin, which are also effective when it comes to extending shelf life. An unsulfated C-terminal fragment of 21 amino acids in hirudin, N-acetylhirudin45.65, inhibits coagulation formation in vitro. In addition, several other smaller, unsulfated peptides corresponding to the C-terminal 11 or 12 amino acids of hirudin (residues 55-65 and 54-65) have also shown good activity in inhibiting clot formation in vitro [J. Krstenansky et al., "Antithrombin Properties of C-terminus of Hirudin Using Synthetic Unsulfated N-acetyl-hiru-din45.65", FEBS Lett, 211, pp. 10-16 (1987)]. However, such peptide fragments have not been completely satisfactory when it comes to dissolving blood clots in ongoing therapeutic courses due to low activity. For example, N-acetyl-hirudin45.65 has a specific activity that is four orders of magnitude lower than natural hirudin.

In addition to catalyzing the formation of a fibrin clot, thrombin has several other bioregulatory roles [J. W. Fenton, II, "Thrombin Bioregulatory Functions", Adv. Clin. Enzvmol., 6, pp. 186-93 (1988)]. E.g. thrombin directly activates platelet aggregation and release reactions. This means that thrombin plays a central role in acute platelet-dependent thrombosis [S.R. Hanson and L.A. Harker, "Interruption of Acute Platelet-Dependent Thrombosis by the Synthetic Ant i thrombin D-Phenylalanyl-L-Prolyl-L-Arginylch-loromethylketone", Proe. Nati. Acad. Pollock. USA, 85, pp. 184-88 (1988)]. Thrombin can also activate an inflammatory response directly by stimulating the synthesis of platelet-activating factor (PAF) in endothelial cells [S. Prescott et al., "Human Endothelial Cells in Culture Produce Platelet-Activating Factor (l-alkyl-2-acetyl-sn-qlycero-3-phospho-choline) When Stimulated With Thrombin", Proe. Nati. Acad. Pollock. USA, 81, pp. 3534-38 (1984)]. PAF is exposed on the surface of endothelial cells and serves as a ligand for neutrophil adhesion and subsequent degranulation [G.M. Vercolletti et al., "Platelet-Activating Factor Primes Neutrophil-Mediated Endothelial Damage", Blood, 71, pp. 1100-07 (1988)-]. Alternatively, thrombin can promote inflammation by increasing vascular permeability which can lead to edema [P.J. Del Vecchio et al., "Endothelial Monolayer Permeability To Macromolecules", Fed. Proe., 46, pp. 1511-15 (1987)] . Reagents that block the active site of thrombin, such as hirudin, interrupt the activation of platelets and endothelial cells [CL. Knupp, "Effect of Thrombin Inhibitors in Thrombin-Induced Release and Aggregation", Thrombosis Res., 49, pp. 23-36 (1988)].

Thrombin has also been implicated in promoting cancer, based on the ability of its natural cleavage product, fibrin, to serve as a substrate for tumor growth [A. Falanga et al., "Isolation and Characterization of Cancer Procoagulant": A Cysteine Proteinase from Malignant Tissue", Biochemistry, 24, pp. 5558-67 (1985); S.G. Gordon et al., "Cysteine Proteinase Procoagulant From Amnion-Chlorion" , Blood, 66, pp. 1261-65 (1985)]; and A. Falanga et al., "A New Procoagulant In Acute Leukemia", Blood, 71, pp. 870-75 (1988)]. And thrombin has been implicated in neurodegenerative diseases due to its ability to induce neurite retraction [D. Gurwitz et al., "Thrombin Modulates and Reverses Neuroblastorna Neurite Outgrowth", Proe. Nati. Acad. Sei. USA, 85, pp. 3440-44 ( 1988)] Therefore, the ability to regulate the in vitro activity of thrombin has many important clinical implications.

Despite advances to date, there is still a need for a molecule that effectively inhibits thrombin function in clot formation, platelet activation, and various other thrombin-mediated processes, and that can be produced cheaply and in commercially affordable quantities.

Summary of the invention

The present invention solves the problems stated above by producing molecules that mimic the action of hirudin by binding to both the low-affinity anion-binding outward site (ABE) and the catalytic site of 6-thrombin. These molecules are more potent than hirudin, and therefore they can be administered to patients in doses that are relatively lower than the doses required in hirudin-based therapeutic regimens. The molecules produced according to this invention can be used in compositions and methods that inhibit any thrombin-generated or thrombin-associated function or process. These molecules can also be used in compositions and methods for ex vivo imaging, for the storage and treatment of extracorporeal blood and for coating invasive equipment. And the molecules of the invention can be administered to a patient together with a fibrinolytic agent to increase the activity of a given dose of that agent, or to lower the dose of the agent required for a given effect, such as dissolving a blood clot.

Due to their high potency and the fact that they can be produced by chemical synthesis techniques, the molecules in question can be produced cheaply in commercially affordable quantities. Furthermore, because the molecules produced according to the present invention are significantly smaller than hirudin, they are less likely to stimulate an unwanted immune response in patients treated with them. Accordingly, the use of these thrombin inhibitors is not limited to the treatment of acute disease. These molecules can also be used in therapy for chronic thromboembolic diseases, such as arteriosclerosis and restenosis after angioplasty. The molecules produced according to the present invention can also be used for a number of other purposes instead of natural or recombinant hirudin.

As will be understood from the following description, the molecules produced according to the present invention are useful in the treatment and prevention of various diseases which are associated with unwanted effects of thrombin, as well as for diagnostic purposes.

Brief description of the drawings

Figure 1 shows an autoradiogram of an SDS-polyacrylamide gel showing the binding of DNFB- [<35>S]-Sulfo-Tyr63<h>irudin54,64

to human 6-thrombin in the presence or absence of Sulf o-Tyr63-N-acetyl-hirudin53.64 .

Figure 2 shows a three-dimensional model of human 6-thrombin. Figure 3, preparation A, shows the effects of Hirulog-8 and Sulfo-Tyr63hirudin53,64 on the cleavage of Spectrozyme TH by human 6-thrombin. Figure 3, Exhibit B, shows a Lineweaver-Burke plot of the cleavage of Spectrozyme TH by human 6-thrombin in the presence or absence of either Hirulog-8 or Sulfo-Tyr63hirudin53_64 - Figure 4 shows the effect of different concentrations of Hirulog-8, hirudin or Sulfo-Tyr63-N-acetyl-hirudin53_64 on the activated partial thromboplastin time in normal human serum. Figure 5, preparation A, shows the time course of the cleavage of different concentrations of Hirulog-8 by human thrombin. Figure 5, preparation B, shows the relationship between the Hirulog-8 concentration and the duration of the inhibition of Spectrozyme TH hydrolysis by human 6-thrombin. Figure 6 shows the effect of the link length of the thrombin inhibitors prepared according to the present invention on the inhibition of the thrombin-catalyzed hydrolysis of Spectrozyme

TH.

Figure 7 shows the inhibitory effect of different concentrations of Hirulog-8 or Sulfo-Tyr63-N-acetyl-hirudin53,64 on the modification of thrombin by <14>C-DFP. Figure 8 shows the in vivo effect of varying doses of Hirulog-8 on APTT in baboons. Figure 9 shows the comparative inhibitory effects of Hirulog-8 or heparin on the hydrolysis of fibrinogen by soluble or clot-bound thrombin. Figure 10 shows the in vivo effects of different doses of Hirulog-8 on the platelet deposition in an endarterectomized segment of baboon aorta. Figure 11 shows the in vivo effects of varying doses of hirulog-8 on the platelet deposition on a segment of a

collagen-coated tube inserted into a baboon.

Figure 12 shows the comparative in vivo effects of heparin, hirudin and Hirulog-8 on platelet deposition on a segment of a collagen-coated tube inserted into a baboon AV bypass. Figure 13 shows the in vivo effects of varying doses of Hirulog-8 on the fibrin deposit on a segment of a collagen-coated tube inserted into a baboon AV bypass. Figure 14 shows the change in APTT over time after intravenous bolus injections of baboons with Hirulog-8. Figure 15 shows changes in APTT over time after subcutaneous injection of baboons with Hirulog-8. Figure 16 shows the comparative in vivo effects of plasminogen tissue activator together with either saline, heparin, hirudin or Hirulog-8 on reperfusion time in a rat model. Figure 17 shows the comparative in vivo effects of plasminogen tissue activator together with either saline, heparin, hirudin or Hirulog-8 on reocclusion time in a rat model. Figure 18 shows the comparative in vivo effects of plasminogen tissue activator together with either saline, heparin, hirudin or Hirulog-8 on APTT in a rat model.

Fig. 19 shows the comparative in vivo effects of plasminogen tissue activator together with either saline, heparin, hirudin or Hirulog-8 on the blood vessel unit in a rat model.

Figure 20 shows the effect of varying doses of Hirulog-8 on bleeding time in a baboon model.

Detailed description of the invention

The following common abbreviations for the amino acids are used in the description and in the claims:

The term "any amino acid" used herein includes the L-isomers of naturally occurring amino acids, as well as other "non-protein" 6-amino acids which are normally used in the field of peptide chemistry when synthetic analogues of naturally occurring amino peptides are produced. The naturally occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyro^ine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, b-carboxyglutamic acid, arginine, ornithine and lysine." Examples of "non-protein" 6-amino acids are norleucine, norvaline, alloisoleucine, homoarginine, tiapro-line, dehydroproline, hydroxyproline (Hyp), homoserine, cyclohexylglycine (Chg), 6-amino-n-butyric acid ( Aba), cyclohexylalanine (Cha), aminophenylbutyric acid (Pba), phenylalanines substituted in the ortho-, meta- or para-position of the phenyl part by one or two of the following; halogen or nitro groups or substituted with a methylenedioxy group; β-2- and 3-thienyl-alanine, S-2- and 3-furanylalanine, S-2-, 3- and 4-pyridylalanine, E-(benzothienyl-2- and 3-yl)alanine, S-(1- and 2-naphthyl)alanine, O-alkylated derivatives of serine, threonine or tyrosine, S-alkylated cysteine, S-alkylated homocysteine, O-sulphate-, O-phosphate- and O -carboxylate esters of tyrosine, 3- and 5-carbonyl-tyrosine, 3- and 5-phosphonyltyrosine, 4-methylsulfonyltyrosine, 4-methylphosphonyltyrosine, 4-phenylacetic acid, 3,5-diiodotyrosine, 3- and 5-nitrotyrosine, "-alkyl-lysine, delta-alkylornithine and the D-isomers of the naturally occurring amino acids.

The compounds referred to here as tyrosine sulfate, Tyr(OS03H) and O-sulfate esters of tyrosine are identical and have the structural formula:

The compounds referred to here as tyrosine sulfonate, Tyr(SO3H), 3-sulfonyltyrosine and 5-sulfonyltyrosine are identical and have the structural formula:

The term "patient" used in this application refers to any mammal, especially man.

The term "anionic amino acid" used herein means a meta-, para- or ortho-, mono- or disubstituted phenylalanine, cyclohexylalanine or tyrosine containing a carboxyl, phosphoryl or sulfonyl part, as well as S-alkylated cysteine, S-alkylated homocysteine, b-carboxyglutamic acid,

~-alkyl-lysine, delta-alkyl-ornithine, glutamic acid and aspartic acid. Examples of anionic amino acids are phosphothreonine, phosphoserine, phosphotyrosine, 3-, 4- or 5-sulfotyrosine, 3-methyl-phosphonyltyrosine and 3-methyl-sulfonyltyrosine.

The terms "catalytic site", "active site" and "active site pocket" as used herein refer to any or all of the following sites in thrombin: the substrate binding site or "S^ site; the hydrophobic binding site or "oil" site; and the site where the cleavage of the substrate is actually performed (the "charge transfer site").

The term "N0™" used herein refers to the side chain nitrogen of ornithine. The designation "N<9>" means any side chain nitrogen of arginine. The designation "N<6>" refers to the 6-amino group in an amino acid. And the term "psi" as used in the specification and claims refers to the replacement of an amide bond with the atoms indicated in parentheses, according to the nomenclature described in J. Rudinger, in Drug Design, Vol. II, E.J. Ariens, ed., Academic Press, New York, p. 319 (1971).

The term "backbone chain" as used herein refers to the part of a chemical structure that constitutes the smallest number of consecutive bonds that can be traced from one end of the chemical structure to the other. The atomic components that make up a backbone chain can consist of any atom capable of forming bonds with at least two other atoms.

For example, each of the following chemical structures is characterized by a backbone chain of 7 atoms (the atoms that make up the backbone chain are indicated in bold type):

The term "calculated length" used in this application refers to a predicted measure obtained by summing the bond lengths between the atoms that make up the backbone chain. Bond lengths between two given atoms are well known in the art [see e.g. CRC Handbook of Chemistry and Physics, 65th Edition, R.C. Weist, ed., CRC Press, Inc., Boca Raton, FL, pp. F-166-70 (1984)].

The present invention relates to the production of molecules that bind to and inhibit thrombin. These molecules are characterized by three domains: a catalytic site-directed portion ("CSDM"), a linker region, and an anion-binding outwardly site-associating portion ("ABEAM").

The first domain, CSDM, binds to the catalytic site in thrombin, which is located at or near approx. Ser-195 and inhibits or delays the amidolytic or esterolytic activity of thrombin. Preferably, the CSDMs are selected from one of three general groups: Those that bind reversibly to thrombin and are slowly cleaved, those that bind reversibly to thrombin and cannot be cleaved, and those that bind irreversibly to thrombin. Reversible inhibitors bind to the active site in thrombin by non-covalent interactions, such as ionic bonds, hydrophobic interactions or hydrogen bonding. Irreversible CSDMs form covalent bonds with thrombin.

According to a preferred embodiment, the CSDM as preparation, which binds reversibly to thrombin and is slowly cleaved, has the formula:

X-Ai-Aa-Aj-Y,

where X is hydrogen or a backbone chain consisting of 1-35 atoms, A1 is Arg, Lys or Orn, A2 is a non-amide bond, A3 is a backbone chain consisting of from 1-9 atoms, and Y is a bond.

The non-amide bond component of this embodiment can be formed by chemically modifying an amide bond. This can be achieved by methods that are well known in the art. [M. Szelke et al., "Potential New Inhibitors of Human Renin", Nature, 299, pp. 555-57 (1982); D. H. Coy et al., "Facile Solid Phase Preparation of Proteins Containing the CH2-NH Peptide Bond Isostere and Application to the Synthesis of Somatostatin (SRIF) Octapeptide Analogues", Peptides 1986, D. Theodoropoulos, ed. Walter DeGruyter & Co., Berlin, pp. 143-46 (1978)] . When a non-amidic bond is formed in this way, it is preferred that the chemical modification is carried out before the addition of the dipeptide containing this bond to the other components of the CSDM or to the rest of the thrombin inhibitor molecule. In this way, dipeptide Al-A2-A3 is added at once, in a single synthesis step, to the rest of the molecule.

According to a more preferred embodiment, A1 is Arg and A3 is Pro, D-Pro or Sar. In this embodiment, A2 is a naturally occurring imide bond that is slowly cleaved by thrombin. This avoids having to form the non-amidic bond in advance and means that Ax and A3 can be added to the rest of the molecule sequentially and not all at once.

As indicated above, CSDMs prepared according to this invention can bind irreversibly to thrombin. Examples of irreversible CSDMs include, but are not limited to, general serine proteinase inhibitors, such as phenylmethylsulfonyl fluoride (PMSF), diisopropylfluorophosphate (DFP), tosylprolylchloromethyl ketone (TPCK), and tosyllysylchloromethyl ketone (TLCK); heterocyclic protease inhibitors, such as D-Phe-Pro-Arg-CHCl 2 (PPACK); and transition analogs, such as difluoroketomethylene.

According to another preferred embodiment of the present invention, the non-cleavable, reversible CSDMer formula is prepared

X-C1-C2-A3-Y,

wherein Cx is a derivative of Arg, Lys or Orn, comprising a carboxylate moiety that is reduced or displaced from a-

the carbon with a backbone chain of from 1 to 10 atoms; C2 is a non-cleavable bond; and X, Y, and A2 are as previously defined. Examples of Cx components are S-homoarginine; arginine containing a reduced carboxylate moiety, such as Arg [p_siCH2NH] ; fi-homolysin and S-homoornithine.

Other non-cleavable, reversible CSDMs that can be used in the thrombin inhibitors prepared according to this invention are benzamidine, DAPA, NAPAP and argatroban (argipidine).

For those thrombin inhibitors that have CSDM regions characterized by an A2 or C2 bond, the term "P-1-Pj/" sequence used herein means the two chemical structures connected by said bond.

The X component of CSDM, which does not participate by actually binding to the catalytic site, can be of unlimited length and variable composition. But for practical purposes and reduced costs of the synthesis, X is preferably characterized by a backbone chain consisting of from 1 to 35 atoms and which does not exceed a calculated length of 3 6 Å. It is preferred that X is a peptide, most preferably D-Phe- Pro. This most preferred embodiment allows the X component to fit into a depression in thrombin located near the active site. [S.Bajusz et al., "Inhibition of Thrombin and Trypsin by Tripeptide Aldehydes", Int. J. Peptide Protein Res., 12, pp. 217-21 (1978)]; C. Kettner et al., "D-Phe-Prp-Arg-CH 2 Cl - A Selective Affinity Label for Thrombin", Thromb. Res., 14, pp. 969-73 (1979)]. This means that the CSDM component and therefore the molecules produced according to the present invention can be bound to thrombin with an advantageously high degree of affinity and optimal specificity.

The second component of the thrombin inhibitors produced according to this invention is a linking region. Because the role of this part of the molecule is to provide a bridge between CSDM and ABEAM, it is the linker's length, and not its structure, that is of primary importance. The calculated length of the backbone chain that characterizes the linker must be at least approx. 18 Å - the distance between the catalytic site and the anion-binding outward-facing site in thrombin - and less than approx. 42 Å.

The backbone chain of the linker can consist of any atom capable of bonding to at least two other atoms. Preferably, the backbone chain consists of any chemically possible combination of atoms selected from oxygen, carbon, nitrogen and sulfur. One skilled in the art is aware that combination of the above backbone chain atoms falls within the required length, based on known distances between different bonds [see e.g. RT Morrison and R.N. Boyd, Organic Chemistry, 3rd Edition, Allyn and Bacon, Inc., Boston, Massachusetts (1977)]. According to a preferred embodiment, the linker is a peptide consisting of the amino acid sequence Gly-Gly-Gly-Asn-Gly-Asp-Phe. Preferably, the amino acid bound to the ABEAM component is Phe.

The third domain in the thrombin inhibitors produced according to this invention is the ABEAM which binds to the anion-binding outward facing site in thrombin. Preferably ABEAM has the formula

W-B1~B2 — B3—B^ — Bg — Bg — B-j—Bq — Z ;

where W is a bond; B1 is an anionic amino acid; B2 is any amino acid; B3 is Ile, Val, Leu, Nie or Phe; B4 is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4-carboxylate, Sar, any N-methylamino acid or D-Ala; B5 is an anionic amino acid; B6 is an anionic amino acid; B7 is a lipophilic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nie, Ile, Val, Cha, Pro, or a dipeptide consisting of one of these lipophilic amino acids and any amino acid; B8 is a bond or peptide consisting of 1 to 5 residues of any amino acid; and Z is a carboxy terminal residue selected from OH, C 1 -C 8 alkoxy, amino, mono- or di-(C 1 -C 4 )alkyl substituted amino or benzylamino.

Peptides homologous to the carboxy-terminal portion in hirudin have been shown to bind to the anionic outward binding site on thrombin [co-pending US patent application

314,756 and J.M. Maraganore et al., "Anticoagulant Activity of Synthetic Hirudin Peptides", J. Biol. Chem., 264, pp. 8692-98 (1989)].

According to a preferred embodiment, ABEAM is homologous to amino acids 56-64 of hirudin, i.e. Bx is Glu, B2 is Glu, B3 is Ile, B4 is Pro, B5 is Glu, B6 is Glu, B7 is Tyr-Leu, Tyr (SO 3 H)-Leu or Tyr (OSO 3 H)-Leu, or (3-,5-diiodoTyr)-Leu, B 8 is a bond, and Z is OH. It should be noted that natural hirudin contains Tyr(OS03H) at position 63. But carboxy termini containing Tyr(SO3H) have exactly the same anticoagulant activity as peptides containing the natural Tyr(OS03H) [see co-pending US patent application 314,756].

Other ABEAM components may include those parts of any molecule that bind to the anionic binding site in thrombin. These include amino acids 1675-1686 in Factor V, amino acids 272-285 of platelet glycoprotein Ib, amino acids 415-428 of thrombomodulin, amino acids 245-259 of prothrombin Fragment 2 and amino acids 30-44 of the fibrinogen Aa chain. In addition, the ABEAM component may be selected from any of the hirudin peptide analogs described by J.L. Krstenansky et al., "Development of MDL-28,050, A Small Stable Antithrombin Agent Based On A Functional Domain of the Leech Protein, Hirudin", Thromb. Haemostas., 63, pp. 208-14

(1990).

The preferred thrombin inhibitors produced according to this invention are called hirulogers, and they are described in the following examples. The most preferred hirulogs are Hirulog-8, Hirulog-12, Hirulog 18a, Hirulog 18b and Hirulog-33. Hirulog-8, -12 and -33 are reversible thrombin inhibitors that cleave slowly. Hirulog-18a and -18b are reversible inhibitors that do not cleave.

The thrombin inhibitors produced according to the present invention can be synthesized using various techniques which are well known in the art. These include enzymatic cleavage of natural or recombinant hirudin, recombinant DNA techniques, solid phase peptide synthesis, liquid phase peptide synthesis, organic chemical synthesis techniques or a combination of these techniques. The choice of synthesis technique will of course depend on the composition of the particular inhibitor. In a preferred embodiment according to this invention, the thrombin inhibitor is completely peptidic and is synthesized by solid-phase peptide synthesis techniques, liquid-phase peptide synthesis techniques or a combination thereof which constitutes the most cost-effective method for producing commercial quantities of these molecules .

When "non-protein" amino acids occur in the throbin inhibitor molecule, they can either be added directly to the growing chain during peptide synthesis or produced by chemical modification of the fully synthesized peptide, depending on the nature of the desired "non-protein" amino acid. Those skilled in the art of chemical synthesis will be aware of which "non-protein" amino acids can be added directly and which must be synthesized by chemically modifying the complete peptide chain after peptide synthesis.

The synthesis of these thrombin inhibitors according to the invention, which contain both non-amino acids and peptide parts, is advantageously obtained by a mixed heterologous/solid phase technique. This technique involves solid-phase synthesis of all or most of the peptide part of the molecule, followed by the addition of the non-amino acid-containing components that are synthesized by liquid-phase techniques. The non-amino acid can be linked to the peptidic part via solid-phase or liquid-phase methods. Similarly, any remaining peptidic moiety can also be added via solid-phase or liquid-phase methods.

The molecules produced according to the present invention exert powerful anticoagulant activity. This activity can be measured in vitro using any conventional technique. Preferably, a determination of the anticoagulant activity involves direct determination of the thrombin-inhibiting activity of the molecule. Such techniques measure the inhibition of thrombin-catalyzed cleavage of colorimetric substrates or, more preferably, the increase in thrombin times or the increase in activated partial thromboplastin times in human plasma. The latter examination measures factors in the "internal" coagulation process. Alternatively, the assay may use purified thrombin and fibrinogen to measure the inhibition of the release of fibrinopeptides A and B by radioimmunoassay or ELISA.

The antiplatelet activity of the molecules of this invention can also be measured by any of a number of conventional platelet assays. Preferably, the examination will measure the change in the degree of aggregation of platelets or the change in the release of a secretion component from the platelets in the presence of thrombin. The former can be measured in an aggregometer. The latter can be measured using RIA or ELISA techniques that are specific for the secreted component.

The molecules produced according to the present invention are useful in compositions, combinations and methods for the treatment and prophylaxis of various diseases attributed to thrombin-mediated and thrombin-associated functions and processes. These include myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, restenosis after arterial damage or cardiological interventional procedures, acute or chronic arteriosclerosis, edema and inflammation, various cell regulatory processes (e.g. secretion, shape changes, proliferation ), cancer and metastasis, and neurogenerative diseases.

The thrombin inhibitors produced according to the present invention can be formulated using conventional methods to form pharmaceutically useful compositions, such as the addition of a pharmaceutically acceptable carrier. These compositions and methods of using them can be used to treat or prevent thrombotic diseases in a patient.

The thrombin inhibitors produced according to the invention can be used in combinations, compositions and methods to treat thrombotic diseases and to reduce the dosage of a thrombolytic agent which is necessary to create reperfusion or prevent reocclusion in a patient. In addition, the thrombin inhibitors produced according to this invention can be used in combinations, compositions and methods to reduce the reperfusion time or increase the reocclusion time in a patient who is treated with a thrombolytic agent. These combinations and compositions comprise a pharmaceutically effective amount of a thrombin inhibitor prepared according to the present invention and a pharmaceutically effective amount of a thrombolytic agent.

In these combinations and compositions, the thrombin inhibitor and the thrombolytic agent act in a complementary manner to dissolve blood clots, resulting in decreased reperfusion times and increased reocclusion times in the patients treated with them. The thrombolytic agent specifically dissolves the clot, while the thrombin inhibitor prevents newly exposed, clot-enclosed or clot-bound thrombin from regenerating the clot. The use of thrombin inhibitors in combinations and compositions allows one to advantageously administer a thrombolytic reagent in amounts previously considered to be too low to produce thrombolytic effects if given alone. In this way, some of the unwanted side effects associated with the use of thrombolytic agents, such as bleeding complications, are avoided.

Thrombolytic agents that can be used in the combinations and compositions are those known in the art. Such agents include, but are not limited to, tissue plasminogen activator purified from natural sources, recombinant tissue plasminogen activator, streptokinase, urokinase, prourokinase, isolated streptokinase plasminogen activator complex (ASPAC), animal salivary gland plasminogen activators, and known biologically active derivatives of all of the above.

The term "combination" as used herein includes a single dosage form containing at least one thrombin inhibitor prepared according to this invention and at least one thrombolytic agent; a multiple dosage form, wherein the thrombin inhibitor and the thrombolytic agent are administered separately but simultaneously; or a multiple dosage form where the two components are administered separately but sequentially. With sequential administration, the thrombin inhibitor can be given to the patient over a period of time varying from approx. 5 hours before until approx. 5 hours after the administration of the thrombolytic agent. Preferably, the thrombin inhibitor is administered to the patient during a period of from 2 hours before to 2 hours after administration of the thrombolytic agent.

Alternatively, the thrombin inhibitor and the thrombolytic agent may be in the form of a single, conjugated molecule. Conjugation of the two components can be achieved by standard cross-linking techniques which are well known in the art. The single molecule can also take the form of a recombinant fusion protein, if both the thrombin inhibitor and the thrombolytic agent are peptidic.

Various dosage forms can be used to administer the compositions and combinations mentioned above. These include, but are not limited to, parenteral administration, oral administration and topical application. The compositions and combinations can be administered to the patient in any pharmaceutically acceptable dosage form, including forms that can be administered to a patient intravenously as a bolus or as a continuous infusion, intramuscularly - including paravertebral and periarticular subcutaneously, intracutaneously, intraarticularly, intrasynovially, intrathecally, intralesionally , periosteally or by oral, nasal or topical route. Such compositions and combinations are preferably adapted for topical, nasal, oral and parenteral administration, but preferably they are formulated for parenteral administration.

Parenteral compositions are most preferably administered intravenously, either in bolus form or as a constant infusion. If the thrombin inhibitor is used as an antiplatelet compound, constant infusion is preferred. If the thrombin inhibitor is used as an anticoagulant, a subcutaneous or intravenous bolus injection is preferred. For parenteral administration, liquid unit dosage forms are prepared, which contain a thrombin inhibitor prepared according to the present invention and a sterile carrier material. The thrombin inhibitor can be either suspended or dissolved, depending on the nature of the carrier and the nature of the particular thrombin inhibitor. Parenteral compositions are usually prepared by dissolving the thrombin inhibitor in a carrier substance, possibly together with other substances, and filter-sterilizing before filling into suitable bottles or ampoules and sealing. Preferably, auxiliary substances, such as local anaesthetics, preservatives and buffers are also dissolved in the carrier substance. The compositions can then be frozen and lyophilized to improve stability.

Parenteral suspensions are prepared in essentially the same way, except that the active component is suspended rather than dissolved in the carrier substance. Sterilization of the compositions is preferably achieved by exposing them to ethylene oxide before suspending them in the sterile carrier substance. Advantageously, a surfactant or wetting agent is added to the composition to facilitate uniform distribution of its constituents.

Tablets and capsules for oral administration may contain conventional excipients, such as binders, fillers, diluents, tableting agents, lubricants, solvents and wetting agents. The tablet can be coated according to methods known in the art. Suitable fillers which can be used include cellulose, mannitol, lactose and other similar agents. Suitable disintegrants include, but are not limited to, starch, polyvinylpyrrolidone, and starch derivatives, such as sodium starch glycolate. Suitable lubricants include e.g. magnesium stearate. Suitable humectants include sodium lauryl sulfate.

Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for dissolution with water or another suitable carrier before use. Such liquid preparations may contain conventional additives. These include suspending agents, such as sorbitol, syrup, methyl cellulose, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel or hydrogenated edible fats, emulsifiers including lecithin, sorbitan monooleate, polyethylene glycols or acacia, non-aqueous carriers, such as almond oil, fractionated coconut oil and oil-like esters, and preservatives, such as methyl or propyl p-hydroxybenzoate or sorbic acid.

Compositions formulated for topical administration can e.g. be in the form of aqueous jelly, oily suspension or emulsified ointment.

The dosage and dose rate of the thrombin inhibitor will depend on several factors, such as the size of the patient, the specific pharmaceutical composition used, the purpose of the treatment, i.e. therapy or prevention, the nature of the thrombotic disease to be treated, and the judgment of the attending physician.

A preferred pharmaceutically effective daily dose of the thrombin inhibitor prepared according to this invention is between approx. 1//g/kg body weight of the patient to be treated and approx. 5 mg/kg body weight. In combinations containing a thrombolytic agent, a pharmaceutically effective daily dose of the thrombolytic agent is between about 10% and 80% of the conventional dosage range. The "conventional dosage range" of a thrombolytic agent is the daily dosage used when this agent is used in monotherapy.

[Physician's Desk Reference 1989, 43rd Edition, Edward R. Barnhart, Publisher]. This conventional dosage range will of course vary, depending on the thrombolytic agent used. Examples of conventional dosage ranges are as follows: urokinase - 500,000 to 6,250,000 units/patient, streptokinase - 140,000 to 2,500,000 units/patient, tPA - 0.5 to 5.0 mg/kg body weight, ASPAC

- 0.1 to 10 units/kg body weight.

Most preferably, the therapeutic and prophylactic compositions comprise a dosage of between approx. 10 /xg/kg body weight and approx. 500 µg/kg body weight of the thrombin inhibitor. Most preferred combinations comprise the same amount of thrombin inhibitor and between approx. 10% and 79% of the conventional dosage range of a thrombolytic agent. It should also be understood that a daily pharmaceutically effective dose of either the thrombin inhibitors or the thrombolytic agent present in the combinations according to the invention may be less or greater than the specific ranges mentioned above.

When an improvement in the patient's condition has occurred, a maintenance dose of a combination or composition as mentioned above is administered, if necessary. Thereafter, the dosage or the frequency of administration, or both, as a function of the symptoms, can be reduced to a level where the improved condition is maintained. When symptoms have been alleviated to the desired level, treatment should cease. However, patients may need intermittent treatment if the disease symptoms reappear.

According to an alternative embodiment, the thrombin inhibitors can be used in compositions and methods for coating the surface of invasive equipment, resulting in a lower risk of clot formation or platelet activation in patients receiving such equipment. Surfaces that can be coated with the compositions include e.g. prostheses, artificial valves, vascular grafts, stents and catheters. Methods and compositions for coating this equipment are known to those skilled in the art. These include chemical cross-linking or physical adsorption of the thrombin inhibitor-containing compositions to the surface of the equipment.

According to a further embodiment, thrombin inhibitors can be used for ex vivo thrombus in a patient. In this embodiment, the thrombin inhibitor is labeled with a radioisotope. The choice of radioisotope is based on a number of well-known factors, e.g. toxicity, biological half-life and detectability. Preferred radioisotopes include, but are not limited to <1>25T, 123I and <11>1In. Techniques for labeling the thrombin inhibitors are well known in the art. Most preferred is the radioisotope 123I, and the labeling is achieved using a <123>I-Bolton-Hunter's reagent. The labeled thrombin inhibitor is administered to a patient and allowed to bind to the thrombin present in a clot. The clot is then observed by means of well-known detection means, such as a camera capable of detecting radioactivity connected to a computer imaging system. This technique also gives an image of

platelet-bound thrombin and meizothrombin.

The effectiveness of the thrombin inhibitors produced according to the invention for the treatment of tumor metastases is confirmed by the inhibition of metastatic weight. This is based on the presence of a procoagulant enzyme in certain cancer cells. This enzyme activates the conversion of factor X to factor Xa in the coagulation cascade, resulting in fibrin deposition, which in turn serves as a substrate for tumor growth. By inhibiting fibrin deposition by inhibiting thrombin, the molecules produced according to the present invention serve as effective antimetastatic tumor agents. Examples of metastatic tumors that can be treated with the thrombin inhibitors produced according to this invention include, but are not limited to, carcinoma of the brain, carcinoma of the liver, carcinoma of the lungs, osteo-carcinoma and neoplastic plasma-cell carcinoma.

The above-described thrombin inhibitors can also be used to inhibit thrombin-induced endothelial cell activation. This inhibition involves suppression of the synthesis of platelet-activating factor (PAF) by endothelial cells. This has an important application in the treatment of diseases characterized by thrombin-induced inflammation and oedema, which is believed to be mediated by PAF. Such diseases include, but are not limited to, respiratory distress syndrome in adults, septic shock, septicemia and reperfusion injury.

Early stages of septic shock include discrete, acute inflammatory and coagulopathic responses. It has previously been shown that injection into baboons with a lethal dose of live EL. coli leads to a clear reduction in neutrophil counts, blood pressure and hematocrit. Changes in blood pressure and hematocrit are partly due to the formation of a disseminated intravascular coagulum (DIC), which has been shown to have parallel consumption of fibrinogen [F. B. Taylor et al., "Protein C Prevents the Coagulopathic and Lethal Effects of Escheveria coli infusion in the Baboon", J. Clin. Invest., 79, pp. 918-25 (1987)]. Neutropenia is due to the severe inflammatory response caused by septic shock, which results in marked increases in tumor necrosis factor levels. The thrombin inhibitors produced according to the invention can be used in compositions and methods to treat or prevent DIC in septicemia and other diseases.

The amount or concentration of thrombin inhibitor in extracorporeal leaf treatment compositions is based on the volume of blood to be treated, or more preferably, its thrombin content. Preferably, an effective amount of a thrombin inhibitor is prepared according to the invention to prevent coagulation in extracorporeal blood from about l^g/60 ml extracorporeal blood to approx. 5 /xg/60 ml extracorporeal blood.

The thrombin inhibitors can also be used to inhibit clot-bound thrombin, which is thought to contribute to clot growth. This is particularly important, because commonly used antithrombin agents, such as heparin and low molecular weight heparin, are ineffective against clot-bound thrombin.

Finally, the thrombin inhibitors produced according to this invention can be used in compositions and methods for the treatment of neurodegenerative diseases. Thrombin is known to cause neurite retraction, a process that indicates a rounding of the shape of brain cells and is implicated in neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.

In order for the described invention to be better understood, the following examples are given. It should be understood that these examples are given for illustrative purposes only and are not to be construed as limiting this invention in any way.

Example 1

Synthesis of Sulfo-Tyr63-N-acetyl-hirudin^-g,] Sulfo-Tyr63-N-acetyl-hirudin54.64 has the amino acid formula: H-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu- Tyr(SO3)-Leu-OH. We prepared this peptide by solid phase peptide synthesis using an Applied Biosystems 430 A Peptide Synthetizer (Applied Biosystems, Foster City, CA).

Specifically, we sequentially reacted 0.259 meq of BOC-O-Leu resin (1% DVB resin) with 2 mmol of protected amino acids. After 10 synthesis cycles, we unblocked the peptide and uncoupled it from the DVB resin by treatment with anhydrous HF : p-cresol : ethyl methyl sulfate (10:1:1, v/v/v). The peptide was further purified on a Vydac C18 HPLC reverse phase column (22 mm x 25 cm), which had been previously equilibrated in 0.1% TFA in water. Before the peptide was loaded onto the column, it was dissolved in 2.0 ml of 0.1% TFA in water. If necessary, extra-Oligere 1 ral 6 H guanidinium chloride was added to the sample to increase solubility. After the addition of the sample, the column was developed with a linear gradient of increasing acetonitrile

(0 - 80%) in 0.1% TFA over 45 minutes at a flow rate of 4.0 ml/min. The exit stream was monitored at 229 nm, and the fractions were collected manually.

The resulting purified peptide was sulfated at the sole tyrosine residue using standard procedures [T. Nakahara et al., "Preparation of Tyroseine-O-[<35>S] Sulfated Cholecystokinin Octapeptide From A Non-Sulfated Precursor Peptide", Anal. Biochem., 154, pp. 194-99

(1986)] . Sulfo-Tyr63-N-acetyl-hirudin54,64 was then purified for others

peptides and reaction components by reverse-phase HPLC using a Vydac C18 column (4.6 x 25 cm) and an Applied Biosystems liquid chromatography system. The column was equilibrated in a 0.1% TFA/water solvent and developed with a linear gradient of increasing acetonitrile concentration

from 0 to 35% over 90 minutes at a flow rate of 0.8 ml/min with a 0.085% TFA-containing solvent. The fractions were measured for absorbance at 214 nm.

Example 2

Cross-linking of human thrombin with Sulfo-Tyr^- Dinitroph luorbenzyl- hirudin54 .64

We prepared Sulfo-Tyr63-dinitrofluorobenzyl-hirudinS4.64 (Sulf o-Tyr63-DNFB-hirudin54.64) by reacting Sulfo-Tyr63-hirudin54.64 (2.0 mg, prepared as in Example 1) with a stoichiometric amount of difluorodinitrobenzene (Pierce Chemical Co., Rockford, IL) in dimethylformamide (DMF) for 18 hours at room temperature. The sample was then subjected to analytical HPLC separation using an Applied Biosystems 150 A liquid chromatography system and a Brownlee RP-300 C8 column (0.46 x 10 cm) to determine the degree of derivatization. The column was equilibrated in 0.1% TFA in water (solvent A) and developed with a 0-50% linear gradient of 0.085% TFA/70% acetonitrile (solvent B) over 45 min and then a 50-100% linear gradient of solvent B for 15 min. We used a constant flow rate of 1.0 ml/min.

The output current was monitored at 214 nm and 310 nm for absorption. Peptide derivatized with the difluoro-nitridebenzene reagent absorbs at 310 nm. We found that the reaction described above produced Sulfo - Tyr63 - DNFB - hirudin54.64 in 15-30% yield. After synthesis, Sulfo-Tyr63-DNFB-hirudin54,64 was stored in the same dimethylformamide solvent at -20°C for up to 1 month.

We reacted a tenfold molar excess of Sulf o-Tyr63-DNFB - hirudin54.64 with human a-thrombin (12.5 mg) for 18 hours at room temperature in phosphate-buffered saline. We determined the degree of cross-linking by analyzing the reaction mixture on an SDS-polyacrylamide gel. SDS-PAGE showed a decrease in the relative mobility of the α-thrombin band, which reflected an increase in molecular weight of 1000-2000 Daltons. This change is consistent with crosslinking of thrombin with Sulfo-Tyr63-DNFB-hirudin54,64 in a single position.

We confirmed that the formation of a covalent complex between Sulf o-Tyr63-DNFB-hirudin54_64 and human thrombin is specific by using [35S]-Sulf o-Tyr63-DNFB-hirudin54.64.

[<35>S]-Sulfo-Tyr63-DNFB-hirudin54.64 was prepared essentially as described above using H2 [^<35>S]04 instead of H2SO4 in the Nakahara sulfation procedure [see also pending US patent applications Nos. 164,178, 251,150, 280,618 and 314,756, and J.M. Maraganore et al., "Anticoagulant Activity of Synthetic Hirudin Peptides'^ J. Biol. Chem., 264, pp. 8692-98 (1989).

We reacted [35S]-Sulfo-Tyr63-DNFB-hirudin54,64 with human α-thrombin, either in the presence or absence of a 5- or 20-fold molar excess (relative to the concentration of thrombin) of Sulfo-Ty<r >63-N-acetyl-hirudin53.64 (prepared as in example 1 with the addition of N-acetyl-asparagine as a final step in the peptide synthesis). After incubation at room temperature for 18 hours, we subjected the mixture to SDA-PAGE and autoradiography. The results (Figure 1) showed that [<35>S]-labeled peptide was incorporated into the band representing thrombin, and that the presence of cold, unlabeled hirudin peptide weakened the magnitude of covalent complex binding to less than 10%. Thus, reaction of Sulfo-Tyr63-DNFB-hirudin54,64 with thrombin results in 1:1 stoichiometric binding of the hirudin peptide at a specific binding site.

To identify the site in thrombin where Sulfo-Tyr63-DNFB-hirudin54.64 binds, thrombin/Sulfo-Tyr63-dinitrobenzyl-(DNB)-hirudin54.64 complex (1.0 mg) was transferred to a Sephadex G-50 column (1.5 x 45 cm), which was equilibrated and developed with 7 M urea, 20 mM Tris, pH 7.5. This chromatography removed all unreacted Sulf o-Tyr63-DNFB-hirudin54_64. A peak containing thrombin/Sulfo-Tyr63-DNB-hirudin54_64 was isolated in the free volume fractions, pooled and reduced by addition of 10 µl of S-mercaptoethanol.

After the reduction, we S-carboxymethylated the complex with iodoacetic acid as described previously [J. M. Maraganore et al., "A New Class of Phospholipases A2 with Lysine in Place of Aspartate-49", J. Bol. Chem., 259, pp. 13839-43 (1984)]. The reduced, S-alkylated protein was then thoroughly dialyzed against 3% acetic acid at room temperature. After the dialysis, we treated the complex with pepsin (2% w/v) for 4 h at 37°C. Peptide fragments of reduced, S-carboxymethylated thrombin/Sulfo-Tyr63-DNB-hirudin54,64 were purified by reverse-phase HPLC using an Aquapore RP-300 C8 column (0.46 x 10 cm). The column was equilibrated in 0.1% TFA in water and developed with a gradient of increasing 0.085% TFA/70% acetonitrile (0-60%) for 80 minutes at a flow rate of 1.0 ml/min. The output stream was monitored for adsorption at both 214 and 310 nm. Fractions of 1.0 ml were collected automatically. HPLC separation of peptide fragments made it possible to resolve a single major peak at both 214 and 310 nm absorbing material. Because of its far UV absorption, this fragment contained the bound Sulf o-Tyr63-DNFB-hirudin54,64.

We then subjected the fragment to automated Edmandegradation with an Applied Biosystems 470A gas phase sequencer equipped with a 900A computer system. Phenylthiohydantoin (PTH) amino acids were analyzed on-line using an Applied Biosystems 12OA PTH analyzer and a PTH-C18 column (2.1 x 220 mm). Shown below is a table of replicate yields from the sequence analysis:

The peptide sequence was found to correspond to residues 144-154 of human α-thrombin [J. W. Fenton, II., "Thrombin Active Site Regions" Semin. Thromb. Hemostasis, 12, pp. 200-08

(1986)]. Peptic cleavages occurred at a Leu-Lys and Val-Leu bond, consistent with the specificity of this enzyme.

During the sequence analysis, the amino acid corresponding to Lys-149 (cycle 10) could not be identified or quantified. This was probably a result of derivatization of

The ~-NH2 group in this amino acid with the dinitrofluorobenzyl moiety in Sulf o-Tyr63-DNFB-hirudin54_64. Thus, Lys-149 is the main site where Sulfo-Tyr63-DNFB-hirudin54_64 reacts with α-thrombin.

Example 3

Preparation of a thrombin inhibitor capable of

block the catalytic site and bind to the outward-directed anionic binding site

Carboxy-terminal hirudin peptides effectively block thrombin-catalyzed fibrinogen hydrolysis but not chromogenic substrate hydrolysis. [J. M. Maraganore et al., J. Biol. Chem.,

264, pp. 8692-98)] . In addition, hirudin peptides do not neutralize thrombin-catalyzed activation of factors V and VIII [J. W. Fenton, II, et al., "Hirudin Inhibition by Thrombin",

Angio. Archive. Biol.. 18, p. 27 (1989)].

Hirudin peptides, such as Sulfo-Tyr63-N-acetyl-hirudirL53_S4, exhibit potent inhibitory effects on thrombin-induced platelet activity in vitro [J. A. Jakubowsky and J. M. Maraganore, "Inhibition of Thrombin-Induced Platelet Activities By A Synthetic 12 Amino Acid Residue Sulfated Peptide (Hirugen)", Blood, p. 1213 (1989)]. Nevertheless, a thrombin inhibitor capable of blocking the active site may be required to inhibit platelet thrombosis in vivo, if activation of Factors V and VIII is the rate-limiting step. This conclusion is supported by results obtained with the irreversible thrombin inhibitor

(D-Phe)-Pro-Arg-CH 2 Cl [S. R. Hanson and L. A. Harker, "Interruption of Acute Platelet-Dependent Thrombosis by the Synthetic Antithrombin D-Phenylalanyl-L-Prolyl-L-Arginyl Chloromethyl Ketone", Proe. Nati. Acad. Pollock. USA, 85, pp. 3184-88 (1988)] and other reversible thrombin inhibitors [J. F. Eidt et al., "Thrombin is an Important Mediator of Platelet Aggregation in Stenosed Canine Coronary Arteries with Endothelial Injury", J. Clin. Invest., 84, pp. 18-27 (1989)-]

Using the above knowledge that the NH2 terminus in hirudin peptides is next to Lys-14 9, we used a three-dimensional model of thrombin (figure 2) [B. Furie et al., "Computer-Generated Models of Blood Coagulation Factor Xa, Factor IXa, and Thrombin Based Upon Structural Homology with Other Serine Proteases", J. Biol. Chem., 257, pp. 3875-82 (1982)] to prepare an agent that: 1) binds to the outward facing anionic binding site in thrombin, and 2) is capable of blocking the active site pockets of thrombin and inhibiting the function of catalytic residues contained therein.

We determined that the minimal distance from ~-NH2 in Lys-149 to the S-hydroxylate in Ser-195 is 18-20 Å. Based on a 3 Å/amino acid residue length, we calculated that at least approx. 4 - 7 amino acids would be necessary to bind a hirudin peptide, such as Sulfo-Tyr63-hirudin53,64, to a region comprising an active site inhibitory structure. The composition of the linker was determined to be glycine. Glycine was chosen to have the greatest possible flexibility in the coupling agent for these preliminary investigations. However, it should be understood that other, more rigid bipolymeric connecting agents can also be used.

We chose the sequence (D-Phe)-Pro-Arg-Pro as the active site inhibitor, because thrombin exerts specificity for Arg as the Pj;-amino acid in the cleavage of substrates. A Pro after Arg gives a bond that is cleaved very slowly by thrombin. We prepared alternative peptides by replacing Pro (after Pj-Arg) with a sarcosyl or N-methyl-alanine amino acid or by chemical reduction of an Arg-Gly cleavable bond.

Example 4

Hirulog- 8 - synthesis

Hirulog-8 has the formula H-(D-Phe)-Pro-Arg-Pro-(Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH. We synthesized

Hirulog-8 by conventional solid phase peptide synthesis using an Applied Biosystems 430 A Peptide Synthetizer. This peptide was synthesized with BOC-L-Leucine-O-divinylbenzene resin. Additional t-BOC amino acids (Peninsula Laboratories, Belmont, CA) used included BOC-Q-2,6-dichlorobenzyltyrosine, BOC-L-glutamic acid (p-benzyl ester), BOC-L-proline, BOC-L- isoleucine, BOC-L-phenylalanine, BOC-L-aspartic acid (S-benzyl ester), BOC-glycine, BOC-L-asparagine, BOC-D-phenylalanine and BOC-L-arginine. To achieve higher yields in the synthesis, the (Gly) 4-linker segment was attached in two cycles with manual addition of BOC-glycyl-

glycine (Beckman Biosciences, Inc., Philadelphia, PA). After the synthesis was completed, the peptide was completely blocked and uncoupled from the divinylbenzene resin by treatment with

anhydrous HF : p_-cresol : ethyl methyl sulfate (10:1:1, v/v/v). After removal from the resin, the peptide was freeze-dried to dryness.

Untreated Hirulog-8 was purified by reverse phase HPLC using an Applied Biosystems 151A liquid chromatography system and a Vydac ■ C18 column (2.2 x 23 cm). The column was equilibrated in 0.1% TFA/water and developed with a linear gradient of increasing acetonitrile concentration from 0 to 80% over 45 min in the 0.1% TFA at a flow rate of 4.0 mL/min. The resulting stream was monitored for absorbance at 229 nm, and fractions were collected manually. We purified 25-30 mg of untreated Hirulog-8 by HPLC and recovered 15-20 mg of pure peptide.

We confirmed the structure of purified Hirulog-8 by amino acid and sequence analyses. Amino acid hydrolysates were prepared by treating the peptide with 6 N HCl in vacuo at 110°C for 24 hours. We then analyzed the hydrolysates by ion exchange chromatography and subsequent ninhydrin derivatization/determination using a Beckman 63 00 automated analyzer. We performed sequence analysis using automated Edman degradation on an Applied Biosystems 470A gas phase sequencer equipped with a Model 900A computer system. Phenylthiohydantoin (PTH) amino acids were analyzed on-line using an Applied Biosystems 12OA PTH analyzer and a PTH-C18 column (2.1 x 220 mm).

Example 5

Synthesis of Hirulog- 9

Hirulog-9 has the formula: H-(D-Phe)-Pro-Arg-D-Pro-(Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu -OH. We synthesized this peptide in the same manner as described in Example 4, using BOC-D-proline (Peninsula Laboratories) at cycle 15 instead of BOC-L-proline. Purification and characterization was carried out as described in Example 4.

Example 6

Synthesis of Hirulog- 10

Hirulog-10 has the formula: H-(D-Phe)-Pro-Arg-Sar-(Gly) 5-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH . The peptide was synthesized as in Example 4 using BOC-sarcosine (Sigma Chemical Co., St. Louis, Mo.) at cycle 16. Purification and characterization was performed as described in Example 4.

Example 7

Synthesis of Hirulog- 11

Hirulog-11 has the formula: H-(D-Phe)-Pro-Arg-Pro-(Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-(3,5- diiodoTyr)-Leu-OH. This peptide was synthesized as in Example 4 using BOC-3,5-diiodo-L-tyrosine (Sigma) at cycle 2. Purification and characterization was performed as described in Example 4.

Example 8

Synthesis of Hirulog- 12

Hirulog 12 has the formula: H-(D-Phe)-Pro-Arg-Pro-(Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr (OS03) -Leu -OH. This peptide is synthesized by reacting 1.0 mg of Hirulog-8 in dimethylformamide (80 µl) with dicyclohexylcarbodiimide solution (1.25 g/ml, 0.007 ml) and concentrated sulfuric acid (0.5 µl) at 0°C in 10 minutes. The reaction is stopped by adding water (1.0 ml).

The reaction mixture can be subjected to reverse phase HPLC using an Applied Biosystems 150A liquid chromatography system and an Aquapore RP-300 C8 column (0.46 x 10 cm). The column is equilibrated in solvent A (0.1% TFA/water) and developed with an increasing concentration of solvent B (0.085% TFA/70% acetonitrile) from 0 to 50% over 45 min at a flow rate of 1.0 ml /my. The output stream is monitored for absorption at 214 nm.

Purified Hirulog-12 is then neutralized to pH 7 by adding 0.1 N NaOH. It is freeze-dried and reconstituted in phosphate-buffered saline.

Example 9

Inhibition of thrombin-catalyzed hydrolysis of a p-nitroanilide synthetic substrate by Hirulog-8

We next analyzed the effects of Hirulog-8 on the human α-thrombin-catalyzed hydrolysis of Spectrozym TH (tosyl-Gly-Pro-Arg-p-nitroanilide; American Diagnostica, New York, NY).

In particular, we measured the initial partial velocities in the presence or absence of Hirulog-8 over a range of substrate concentrations from 2.2 to 22 µM. The thrombin-catalyzed value was monitored in a Cary 19 spectrophotometer at 405 nm and recorded continuously as a function of time. The kinetics were performed at room temperature (25 + 1°C) in a 0.05 M Tris, pH 7.5, 0.1 M NaCl buffer.

For a typical enzyme reaction, 1.0 ml of buffer was added to both the sample and preference cuvettes. Thrombin (3.2 x 10"<9> M, final concentration) and Hirulog-8 (0-4 x 10"<8> M) were added to the sample cuvette prior to the addition of Spectrozym TH (2.2 - 22 /xM). Immediately after adding the substrate, the contents of the sample cuvette were mixed using a plastic pipette. The reaction was monitored spectrophotometrically for 5-15 minutes.

Initial partial rates for each substrate concentration were expressed as mol Spectrozym TH-hydrolyzed/sec/mol thrombin. This was determined during the initial linear phase of the reaction (<.15% total hydrolysis of the substrate) by measuring the rise of the hydrolytic reaction. Linewater-Burke plots were made accordingly by plotting the inverse of the initial velocity against the inverse of the substrate concentration. The results showed that human α-thrombin-catalyzed hydrolysis of Spectrozyme TH had a vmax = 17 m°l hydrolyzed/sec/mol thrombin and a KM value of 1.19 x 10~s M. Figure 3, areas A and B, show that increasing concentrations of Hirulog-8 led to significant dose-dependent increases in the KM value, with small increases in the Vmax value for Spectrozyme TH hydrolysis. Therefore, the inhibition of thrombin-catalyzed turnover of Hirulog-8 was performed by mixed competitive/non-competitive components with respect to the Spectrozym-TH hydrolysis. The Kj value v Hirulog-8 for α-thrombin was determined using the equation:

v max

V

where ( ) is the rise of the thrombin-catalyzed r^ M inhibited

turnover in the presence of Hirulog-8; [Hirulog-8] is the molar " max

V

concentration of peptide; ( ) the thrombin-IXK is uninhibited

catalyzed turnover in the absence of the inhibitor; and KT is the molar inhibition constant for Hirulog-8 with human α-thrombin. The Kj value for Hirulog-8 was calculated to be 1.95 + 0.11 x 10'<9> M.

Example 10

Specificity of Hirulog-8 for the hirudin peptide binding site and the active site in human α-thrombin

Hirulog-8 was synthesized as an analog that binds human α-thrombin via its hirudin peptide binding site while blocking thrombin's catalytic site. We examined the ability of Hirulog-8 to perform these functions in various studies described below.

The kinetics of the Hirulog-8 inhibition of human b-thrombin was studied essentially as described above in Example 9 for human a-thrombin. The p-thrombin catalyzed reaction against Spectrozyme TH showed a Vmax = 7.14 mol hydrolyzed/sec/mol thrombin and KM = 1.1 x 10"<6>M. These results confirm that b-thrombin, a proteolytic form of thrombin, exhibits almost complete catalytic function, although this form essentially lacks coagulant activity [S. D. Lewis et al., "Catalytic Competence of Human A- and p-Thrombins in the Activation of Fibrinogen and Factor XIII", Biochemistrv, 26, pp. 7597-7603 (1987)]. The inhibition of β-thrombin by Hirulog-8 was examined over a range of peptide concentrations from 2.7 x 10"<8> to 6.8 x 10"e

M.

As shown below, Hirulog-8 exerts an increased Kx of 3 orders of magnitude relative to α-thrombin. This high Kx value towards b-thrombin is due to the absence of an intact anion-binding outward facing site (ABE) in p-thrombin [J. W. Fenton, II, et al., "Anion-Binding Exosite of Human a-Thrombin and Fibrinogen) Recognition", Biochemistry, 27, pp. 7106-12

(1988)].

b-Thrombin is formed by proteolysis of the B-chain in a-thrombin at Lys-149 and Arg-78.

The inhibition of human α-thrombin by Hirulog-8 was markedly reduced in the presence of Sulfo-Tyr63-N-acetyl-hirudin53.64 at concentrations of 2.6 x 10"<6>M to 1.29 x 10"<5> M. This is because Sulf o-Tyr63-N-acetyl-hirudin53_64 competes with Hirulog-8 for binding to ABE in thrombin.

This was also shown by addition of phenylmethylsulfonyl-α-thrombin ("PMS-α-thrombin"; 18 nm, final) to reactions of Hirulog-8 with human α-thrombin. The addition of this modified thrombin resulted in a substantial reduction in Hirulog-8's ability to inhibit α-thrombin. PMS-a-thrombin has an intact ABE, but is covalently derivatized at its active site. This modified thrombin sequesters Hirulog-8 in the reaction mixture and therefore reduces the amount of peptide available to inhibit intact, catalytically active human α-thrombin.

We also performed studies on the effect of salt concentrations on the Kl value of Hirulog-8 against thrombin as described above in Example 9. We measured the Kx value in the presence or absence of Hirulog-8 (11.5 x 10"<9> M) in buffers containing 0.1, 0.25 and 0.5 M NaCl As shown in the table below, the inhibition of α-thrombin by Hirulog-8 increased at lower salt concentrations This result confirmed that the action of the highly anionic hirudin peptide moiety in Hirudin -8 with the positively charged site around Lys-149 in thrombin is crucial for Hirulog-8 inhibition in thrombin-catalyzed hydrolysis of Spectrozyme TH.

Example 11

Anticoagulant activity of Hirulog-8: Comparison with

Hirudin and Sulfo- Tyr63- N- acetyl- hirudinrVC^

We studied the anticoagulant activity of Hirulog-8 with pooled normal human plasma (George King Biomedi-cal, Overland Park, KA) and a Coag-A-Mate XC instrument

(General Diagnostics, Organon Technica, Oklahoma City, OK). Activity was monitored using the activated partial thromboplastin time (APTT) test with calcium chloride and phospholipid solutions obtained from the manufacturer. Hirulog-8, hirudin, or Sulf o-Tyr63-N-acetyl-hirudin53_64 was then added to APTT assay wells at a final concentration of 10 to 32,300 ng/ml in a total volume of 25 µl before addition of 100 fj. 1 plasma.

The APTT control (absence of inhibitor) was 29.6 sec (mean, n=8, SEM < 0.5%). Figure 4 shows the results of these dose-dependent investigations. Hirulog-8 was 2 to 3 times more potent than Hirudin and 100 to 150 times more potent than Sulf o-Tyr63-N-acetyl-hirudin53_64. Both Hirulog-8 and hirudin increased the APTT value in plasma to values that were too high to be measured. This is in contrast to Sulf o-Tyr63-N-acetyl-hirudin53„64/ which exerts a saturable dose response in the APTT value of 200-250% compared to the controls [J. M. Maraganore et al., J. Biol. Chem., 264, pp. 8692-98, (1989)-]. This result showed that Hirulog-8 can block the active site of thrombin in plasma, also in vivo in chromogenic studies, in a similar way to hirudin.

Example 12

Inhibition of thrombin-induced platelet activation with Hirulog- 8 Thrombin-induced platelet activation studies are performed at 37°C using a Biodata PO, platelet aggregometer. Platelet-rich plasma (PRP) is obtained from normal, healthy, voluntary persons who have not taken any medication that changes platelet function for at least one week before the examination. PRP is prepared as described by J. A. Jakubowsky et al., "Modification of Human Platelet by a Diet Enriched in Saturated or Polyunsaturated Fat", Atherosclerosis. 31, pp. 335-44 (1978)]. Varying concentrations of Hirulog-8

(0-500 ng/ml in 50 µl of water) is added to 0.4 ml of pre-warmed (37°C) PRP. A minute later we add human

lig a- thrombin to the platelet suspension to a final concentration of 0.2, 0.25 or 0.5 units/ml of the total examination volume. Aggregation is monitored as an increase in light transmission for 5 minutes after the addition of thrombin. We then calculate % inhibition as (%aggregate j onprave) /

(% aggregation control) x 100. This study shows that Hirulog-8 blocks thrombin-induced platelet activation in vitro.

Example 13

Application of Hirulog-8 in thrombus imaging

Hirulog-8 is modified by covalent attachment to a 123i-containing chemical group. Specifically, Hirulog-8 (prepared as in Example 4) is reacted with <123>I-Bolton Hunter Reagent (New England Nuclear, Boston, Mass.) in 0.1 M sodium borate, pH 9.0. The 123 -1 labeled molecule (with a specific activity of >5 iiCi//ug) is then desalted in a Biogel P2 column equilibrated in a phosphate buffered saline.

Ex vivo imaging of experimental thrombi is performed essentially as described by T. M. Palabrica et al., "Thrombus Imaging in a Primate Model with Antibodies Specific for an External Membrane Protein of Activated Platelets", Proe. Nati. Acad. Pollock. USA, 86, pp. 1036-40 (1989). Specifically, imaging is performed in baboons using an external Ticoflex bypass line between the femoral artery and the femoral vein. An experimental thrombus is formed by placing a segment of a precoagulated Dacron graft into the bypass line. 123I-labelled thrombin inhibitor is injected into the venous part of the Tricoflex circuit line. Preceding

serial images are then obtained for 0.5 - 1 hour using an Ohio Nuclear Series 100 Gamma camera with a PDP-ll/34 computer. The kinetics of <123>I-thrombin inhibitor uptake with the graft device and the blood supply is derived from the radionuclide images thus obtained.

The same technique can be used to obtain ex vivo images of a deep venous thrombus caused by stasis in the femoral vein in baboons. Because 123I-Hirulog-8 binds to thrombin with high specificity, precise ex vivo images of thrombi can be obtained by using this molecule. The small size of Hirulog-8 also provides, in contrast to natural hirudin or antibodies to thrombin, the potential where the radioactively labeled thrombin inhibitor will provide images of platelet-bound thrombin and meizothrombin, as well as thrombin occurring in the fibrin coagulate.

Example 14

Antimetastatic activity of thrombin inhibitors

The antimetastatic activity of the thrombin inhibitors of this invention, preferably Hirulog-8, is investigated using sarcoma T241 cells [L.A. Liotta et al., Nature, 284, pp. 67-68 (1980)] and syngeneic C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME). The mice are injected either intra-venously or subcutaneously with 0 - 250 g/kg Hirulog-8, prepared as in example 4, followed by injection with 10<4> - 10<6> T241 tumor cells. After 15 days, the animal is killed, and the lung tumor colonies are quantified. Antimetastatic activity of Hirulog-8 is measured as a percentage reduction in the tumor colonies compared to placebo-treated control mice. Hirulog-8 shows antimetastatic activity in this assay.

Example 15

Inhibition of endothelial cells with a thrombin inhibitor

The ability of the thrombin inhibitor of this invention to prevent thrombin-induced synthesis of the platelet-activating factor (PAF) is investigated using cultured human endothelial cells from the umbilical vein

(HUVEC). The HUVECs are extracted from human umbilical cords by collagenase digestion according to established methods [M. A. Gimborne, Jr., "Culture of Vascular Endothelium", Prog. Hemostat. Thromb., 3, pp. 1-28 (1976)].

The HUVECs are grown to confluence in a 96-well microtiter plate in the presence of [<3>H]-acetate. Cells cultured in this manner elicit [<3>H]-acetyl-PAF, which can be quantified by extraction of HUVEC membrane phospholipids.

Hirulog-8 (0-1 µg/ml) is added to the [ 3 H]-acetate-supplemented HUVECs 1 minute before the addition of thrombin (final concentration 1 U/ml). The cells are incubated for 5 minutes, and the supernatant is then removed. A medium containing 0.1% gelatin, 50 mM acetic acid in methanol (2:1 v/v) is then added to the HUVECs. PAF is then extracted and quantified using conventional techniques [T. M. Mc-Intyre et al., "Cultured Endothelial Cells Synthesize Both Platelet-Activating Factor and Prostacyclin in Response to Histamine, Bradykinin and Adenosine Triphosphate", J. Clin. Invest., 76, pp. 271-80 (1985)]. The IC50 values are then calculated. Hirulog-8 inhibits the synthesis of PAF by HUVEC in this study.

The effect of Hirulog-8 on thrombin-induced polymorphonuclear leukocyte (PMN) adhesion to HUVEC can be shown as follows. The HUVECs are grown to confluence in MEM containing 1% fetal calf serum in 24-well colony plates. The medium is then removed, the cells are washed twice with fresh, serum-free medium and incubated in the same medium for 10-30 minutes at 37°C to remove serum products. PMNs (2.5 x 10<6> in 1 ml), pre-equilibrated at 37°C, are then added to each well. The PMNs are allowed to settle in the HUVEC monolayer for 2 min. Hirulog-8 (5 µg/ml) or saline is added to each well, immediately followed by the addition of α-thrombin (0.1 or 1 U/ml). The cells are incubated for 5 minutes at 37°C, washed twice and then examined by phase contrast microscopy. Adherent PMNs are counted directly. Samples incubated with Hirulog-8 have significantly fewer adherent PMNs than samples treated with saline.

Example 16

Synthesis of Hirulog- 13

Hirulog-13 has the formula: H-(D-Phe)-Pro-Arg-Pro-(Gly) 2-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH . We synthesized, purified and characterized this peptide essentially as described in Example 4, except that only one cycle in BOC-glycylglycine was used to produce the di-glycine segment.

Example 17

Synthesis of Hirulog- 14

Hirulog-14 has the formula: H-(D-Phe)-Pro-Arg-Pro-(Gly) 5-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH . Hirulog-14 was synthesized, purified and characterized using methods described in Example 4, except that one cycle of BOC-glycine addition was used after the two cycles of BOC-glycylglycine addition to prepare the pentaglycine segment.

Example 18

Synthesis of Hirulog- 15

Hirulog-15 has the formula: H-(D-Phe)-Pro-Arg-Pro-(Gly)S-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH . Hirulog-15 was synthesized, purified and characterized using methods described in Example 4, except that three cycles of BOC-glycylglycine addition were used to prepare the hexaglycine segment.

Example 19

Synthesis of Hirulog- 16

Hirulog-16 has the formula: H-(D-Phe)-Pro-Arg-Pro-(Gly)8-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH . Hirulog-16 was prepared, purified and characterized as described in Example 4, except that four cycles of BOC-glycylglycine addition were used to prepare the octaglycine segment.

Example 20

Synthesis of Hiruloq- 17

Hirulog-17 has the formula: H-(D-Phe)-Pro-Arg-Pro-Gly-Gly-Glu-Gly-His-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu- Glu-Tyr-Leu-OH., Hirulog-17 was synthesized essentially as described in Example 4, except that a Gly-Gly-Glu-His-Gly sequence replaced the Gly<4-> segment found in Hirulog-8. This sequence was added to the growing peptide chain by the successive additions of BOC-glycine, BOC-L-histidine, BOC-glycine, BOC-L-glutamic acid and BOC-glycylglycine at cycles 13-17 of the synthesis. Purification and characterization was carried out as described in Example 4.

Example 21

Synthesis of Hiruloq- 18a, - 18b and - 18c

Hirulog-18a has the formula: H-(D-Phe)-Pro-(iS-homoarginine)-(Gly) 5-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu -OH. Hirulog-18b has the formula: H-(D-Phe)-Pro-(S-homoarginine)-Pro-(Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr -Leu-OH. Hirulog-18c has the formula: H-(D-Phe)-Pro-(S-homoarginine)-Val-(Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr -Leu-OH. We synthesized Hirulog-18a by a mixed homogeneous/phase method. Residues 5-20 were prepared by solid-phase synthesis, as described in Examples 4 and 17. The resulting resin-coupled intermediate was reacted with a BOC-S-homoarginine-Gly-protected intermediate, which was synthesized in the multistep reaction core depicted below and described immediately thereafter.

N^-BOC-IvP-Tos-arginine-diazomethyl ketone

We stirred 10 g (13.4 mmol) KP-BOC-KF-Tos-arginine-(Bachem, Torrance, CA) and 2.1 ml (19.1 mmol) N-methylmorpholine (Aldrich, Milwaukee, Wis.) in 100 ml of anhydrous tetrahydrofuran (THF) under argon for 5 minutes at room temperature. The solution was then cooled to -15°C, and 2.8 mL (21.6 mmol) of isobutyl chloroformate (Aldrich) was added. We continued to stir the reaction mixture at -15°C for 5 minutes and then filtered it through a pad of celite/MgSO 4 . Then we added the filtrate to an ice-cooled ethereal solution of diazomethane (150 mM, prepared from 32.4 g of Diazald; Aldrich). The solution was stirred and gradually allowed to reach ambient temperature overnight. The solvent was then removed in vacuo and the residue was dissolved in 200 ml of chloroform. We then washed the organic solution successively with 200 mL saturated NaHCO 3 , followed by 200 mL saturated NaCl, dried it over anhydrous magnesium sulfate, and concentrated it again to an oily residue. The residue was then purified by flash chromatography on a 4 x 17 cm column of silica gel, using a step gradient of acetone in chloroform (10% acetone in 2 1 chloroform, followed by 20% acetone in 3 1 chloroform). Fractions of 25 ml were collected. Aliquots of each fraction were examined by thin-layer chromatography (TLC). Fractions containing the desired product were combined and evaporated to dryness. The product, diazomethyl ketone, was purified as a pale yellow foam (6.54 g) •

N"-BOC-lSF-Tos-£-homoarginine methyl ester

We dissolved the diazomethyl ketone prepared above in 100 ml of anhydrous methanol and refluxed the solution under argon while a solution of silver benzoate catalyst (165 mg in 400 µl of triethylamine) was added dropwise. After 30 minutes, the heated solution was cooled to room temperature, slurried with "Norit", and filtered through celite. The solvent was then removed in vacuo and the oily residue was purified by flash chromatography over silica gel. Elution was achieved with 4 L of 10% acetone in chloroform. The desired product, 13-homoarginine methyl ester, was thus purified as a light brown foam

(6.43 g).

Na<->BOC-ISP-Tos-S-homoarginine

We dissolved all of the above methyl ester in 100 mL methanol and then reacted it with a solution of LiOH (1.48 g in 50 mL water) overnight at room temperature under argon with constant stirring. We removed the methanol in vacuo, dissolved the residue in water and washed it with ethyl acetate. We then added saturated citric acid until the solution reached a pH value of 4. We extracted the resulting carboxylic acid in ethyl acetate. The extraction was repeated at pH 3, and the combined organic phases were dried over magnesium sulfate and concentrated in vacuo. The resulting crude acid was recovered as a white foam (4.9 g). The acid was further purified on a Vydac C18 reverse phase HPLC column, as described in Example 4, except that the exit stream was monitored at 214 nm. After lyophilization of the desired fraction, the product, N^BOC-ISF-Tos-S-homoarginine, was recovered as a white amorphous solid.

A sample of ^-BOC-1SP-Tos-S-homoarginine was hydrolyzed in HF and used as a standard for the amino acid analysis. The retention time of fi-homoarginine was identical to that of arginine, but the intensity of the peak was considerably lower, as expected.

N^BOC-N^Tos-S-homoargininylglycine benzyl ester

We then combined 4.06 g (9.2 mmol) of the above carboxylic acid with 2.04 ml of N-methylmorpholine in 25 ml of anhydrous THF. The mixture was stirred under argon at -5°C. A cooled solution of isobutyl chloroformate (2.4 mL in 25 mL THF) was then added dropwise to the solution over 10 min. After this addition, the reaction mixture was stirred for 12 minutes at -5°C. For Hirulog-18a, we then added a solution of glycine benzyl ester (4.9 g in 40 mL THF; 27.6 mmol), and allowed the reaction mixture to warm to room temperature. The solvent was then removed in vacuo, and the resulting residue was dissolved in 100 ml of ethyl acetate. The solution was extracted successively with 100 ml saturated NAHCO 3 and 100 ml saturated NaCl, dried over magnesium sulfate and concentrated in vacuo. The resulting crude dipeptide ester was purified on a 4 x 20 cm silica gel column with a methanol step gradient in chloroform containing 10 drops of NH 4 OH per 100 ml (2 1 1% methanol in chloroform, followed by 3 12% methanol in chloroform). Fractions (25 mL) were collected, examined by TLC, and the fractions containing the product were pooled and the solvent was removed in vacuo. The resulting product, Na<->BOC-Ng<->Tos-S-homoargininyl glycine benzyl ester, was isolated as a white foam (3.9 g).

For Hirulog-18b and -18c, the above reaction was identical, with the exception of the following modifications: For Hirulog-18b, the glycine benzyl ester was replaced with proline benzyl ester, and the reaction was carried out on a 1.8 mmol scale. For Hirulog-18c, the glycine benzyl ester was replaced with valine benzyl ester, and the reaction was performed on a 3.0 mmol scale.

KT-BOC-lSP-Tos-6-homoargininylglycine

The above benzyl ester was dissolved in 50 mL of methanol and hydrogenated at atmospheric pressure over 1.0 g of 10% palladium/carbon for 17 h. The resulting solution was filtered through celite and the solvent was removed in vacuo. The reaction gave 2.9 g of crude Na<->B0C-N<3->Tos-S-homoargininyl glycine, which was purified on a Vydac C18-HPLC column as described above.

The above N 3 BOC-ISF-Tos-S-homoargininyl glycine (1.02 g) was dissolved in 1 ml of anhydrous DMF and cooled in an ice bath. We then added to this solution successively 5.5 ml of 0.5 M hydroxybenztriazole in DMF (Applied Biosystems Inc, Foster City, CA) and 5.5 ml of 0.5 M dicyclohexylcarbodiimide in CH 2 Cl 2 (Applied Biosystems). After 1 hour, the cold suspension of symmetrical anhydride in the dipeptide unit was quickly filtered through a plug of glass wool to remove the dicyclohexylurea.

Meanwhile, a suspension of N-BOC-(Gly)4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-O-PAM (0.2 mmol CH2Cl2) was activated by standard peptide synthesis methods. A Kaiser test on the resulting product indicated a free terminal amino group.

The activated S-homoarginylglycine dipeptide was then coupled to the resin-bound hexadecapeptide. The resulting octadecapeptide was then coupled sequentially with N-BOC-Pro and N-BOC-(D-Phe) using standard coupling methods. The resulting peptide, Hirulog-18a, was purified and characterized as described in Example 4.

A similar procedure was carried out for the synthesis of Hirulog-18b and Hirulog 18c.

Example 22

Synthesis of Hirulog- 19

Hirulog-19 has the formula: H- (D-Phe)-Pro-Arg- [p_siCH2NH] -

(Gly) 5-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH. Residues 4-20 of this peptide were assembled by solid-phase peptide synthetic procedures as described in Example 4. The next residue added, N 2 -BOC-ISP-tosyl-argininal, was prepared as depicted and described below.

Na<->BOC-N<9->Tos-Argininal

ISP-BOC-ISF-Tos-arginine (Bachem Inc.; 10 g) was added to 80 mL of anhydrous THF, and the suspension was cooled to 0-5°C. We then added 1,1'-carbonyldiimidazole (Aldrich; 3.61g) all at once and continued stirring for 20 minutes. The resulting clear solution was partially immersed in a dry ice/acetone bath to maintain a temperature of -20° to -30°C during the dropwise addition of a suspension of lithium aluminum hydride (Aldrich; 1.8 g in 80 mL THF) in during 45 minutes with constant stirring. The mixture was stirred for a further 30 minutes at -20°C and then quenched by the dropwise addition of 63 ml of 2N HCl at -10°C. We filtered the resulting solution through a medium sintered glass funnel and concentrated the resulting filtrate in vacuo.

The resulting crude aldehyde, recovered as a white foam (11.5 g), was suspended in 100 mL of chloroform, washed with water (2 x 50 mL), and the organic layer was then dried over sodium sulfate and concentrated in vacuo. The untreated aldehyde (7.7 g) was dissolved in 100 ml of chloroform and purified by flash chromatography over a 5 x 20 cm flash column containing 350 ml of silica gel (Merck Grade 60, 230-400 mesh, 60 Å). Elution was achieved with a step gradient of 0.5% methanol in 500 ml chloroform, 1% methanol in 1 1 chloroform and 1.5% methanol in chloroform. This procedure gave 8.9 g of Na<->B0C-N<9->Tos-argininal.

N^-BOC-NS-Tos-argininal (258 mL) was then added to the resin-bound (Gly) 5-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-O -PAM under solid-phase reductive alkylation conditions (40 mg sodium cyanoborohydride for 24 hours) using the method described by D. H. Coy et al., "Solid-Phase Synthesis of Peptides" in Peptides, Vol. 8, pp. 119-121 (1978 ). After reaction of the resin-bound peptide with the protected argininal, the peptide synthesis was completed with a cycle of BOC-proline incorporation and a cycle of BOC-(D-phenylalanine) incorporation. After the synthesis was complete, Hirulog-19 was unblocked and decoupled from the resin as described in Example 4.

Hirulog 19 was purified by reverse phase HPLC using an Applied Biosystems 151A liquid chromatography system and an Aquapore C8 column (10 x 22 cm). The column was equilibrated in 1 part 70% acetonitrile/30% water containing 0.85% TFA (Buffer B) and 4 parts water containing 1% TFA (Buffer A). The column was developed with a linear gradient of increasing Buffer B concentration (20-50%) over 120 minutes at a flow rate of 4.0 ml/min. The resulting stream was monitored for absorbance at 214 nm, and fractions were collected manually. Further purification was performed under isocratic conditions using 20% Buffer B/80% Buffer A.

Example 23

Synthesis of Hiruloq- 21

Hirulog-21 has the formula: H-(D-Phe)-Pro-Arg-Pro-(Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu- ( Gly) 2-Lys-OH. Hirulog-21 was synthesized by methods described in Example 4, using the appropriate BOC amino acids. Purification and characterization of Hirulog-21 was achieved by the methods described in Example 4.

Example 24

Synthesis of Hirulog- 25

Hirulog-25 has the formula: H-(D-Phe)-Pro-Arg-Pro-(4-Argininyl-2, 2-difluoro)malonylglycyl-(Gly) 4-Asn-Gly-Asp-Phe-Glu -Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH. The hexadecapeptide, (Gly)4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu, was synthesized as described previously and allowed to be bound to the resin. The next residue, (3-Argininyl-2,2-difluoro)malonyl-glycine, is synthesized in the reaction scheme depicted and described below.

1- r( 2'- Carboethoxy- 1'. 1'- difluoro) etvll

Na<->BOC-N°<rn->benzyl-N°<rn->Cbz-ornithinol

A solution of 3.1 g (7.1 mmol) Na<->BOC-N<orn->benzyl-Norn-Cbz-ornitinal [F. Salituro et al, "Inhibition of Aspartic Proteinases By Peptides Containing Lysine and Onithine Side Chain Analogues of Statins", J. Med. Chem., 30, pp. 286-95

(1987)], and 1.56 mL (9.23 mmol) of ethyl bromodifluoroacetate in anhydrous 15 mL of THF was added over 90 min to a refluxing suspension of 786 mg of Zn powder (Fluka) in 15 mL of THF under argon. After 4 hours of heating under reflux and 2 hours at room temperature, the mixture was cooled and partitioned between 200 ml of ethyl acetate and 200 ml of saturated NaCl/KFS0 4 . The organic phase was isolated, dried over magnesium sulfate and concentrated in vacuo. The resulting oily residue was purified on silica gel using CHCl 3 :methanol (90:10) plus 100 drops/l NH 4 OH as eluent.

1- r( 2'- Carboethoxy- 1'. 1'- difluoro) etvll

Na<->BOC-ornithinol tert-butyldimethylsilyl ether

The resulting compound, 1-[(2'-carboethoxy-1'-1'-difluoro)ethyl]N<a->BOC-N<orn->benzyl-N<orn->Cbz-ornithinol, is then reacted with 5 equivalents of tert-butyldimethylsilyl chloride and 10 equivalents of imidazole in anhydrous DMF at 35°C, according to the method described by E. J. Corey et al., "Protection of Hydroxyl Groups as tertBut<y>ldimethylsilyl Derivatives", J. Amer. Chem. Soc., 94. pp. 6190-91, (1972)]. The orthogonally protected amine is then dissolved in methanol and hydrogenated over Pd(OH)2 at 2 Atm for 18 hours. The catalyst is then removed by filtration, and the filtrate is concentrated in vacuo to give 1-[(2'-carboethoxy-1'-1'-difluoro)-ethyl] Ng-BOC-ornithine-tert-butyldimethylsilyl ether.

1- r( 2 '- Carboethoxysv- 1'. 1'- difluoro) etvll

IST-BOC-ftF-Tos-argininol-tert-butyldimethylsilyl ether

The compound prepared above is then reacted with 6.8 equivalents of both 1-guanyl-3,5-dimethylpyrazole and triethylamine in water at 105°C for 24 hours. The mixture is lyophilized, and the residue is subjected to preparative HPLC as described in example 4. Fractions containing the desired guanidinium compound (examined by TLC) are combined and dried in vacuum. The residue is dissolved in H2O:acetone (1:4), cooled in an ice bath and adjusted to pH 12 with 50% W7V NaOH. To this solution we add a solution of 3 equivalents of paratoluene-sulfonyl chloride in acetone over 60 minutes, while the pH value is kept at 11-12 with NaOH. The solution is allowed to warm to room temperature and stirred overnight. The acetone is then removed in vacuo, and the remaining aqueous solution is washed with ether. The ether layer is removed and back-extracted with saturated NaHCO 3 . The aqueous phases are combined and acidified to pH 3 with 2 N HCl. The resulting acidic solution is extracted twice with ethyl acetate, dried and concentrated in vacuo to give the desired product.

1- f( 2'-Carboxv- 1', 1'- difluoro) etvll

N^BOC-NS<->Tos-argininol

The resulting compound, 1-[(2'-carboethoxy-1'-1'-difluoro)ethyl]N^BOC-ISF-Tos-arquininol-tert-butyldimethylsilyl-ether, is desilylated by treatment with 3 equivalents of tetra-n- butylammonium fluoride in THF at room temperature, as described in E. J. Corey et al., supra. The compound produced by this method is then saponified by treatment with 2.5 equivalents of LiOH in methanol/water at room temperature overnight under argon. The reaction mixture is then washed with ethyl acetate and acidified with citric acid to pH 4. We extract the resulting acid in ethyl acetate, dry the organic phase and concentrate it in vacuo. The untreated acid is then purified on a Vydac '-18

reverse phase HPLC column under the conditions described in Example 4.

1- f( 2'-Carboxv- 1', 1'- difluoro) etvll

N^BOC-KF-Tos-argininone

The alcohol function in the above compound is converted to the ketone by adding an equivalent of pyridinium dichromate in CH2C12 containing 0.5% glacial acetic acid, in the presence of molecular sieves [N. Peet et al., "Synthesis of Peptidyl and Fluoro-methyl Ketones and Peptidyl α-Keto Esters as Inhibitors of Porcine Pancreatic Elastase, Human Neutrophil Elastase, and Rat and Human Neutrophil Cathepsin G", J. Med. Chem., 33, pp. 394-407 (1990)]. After stirring under argon for 15 hours, the reaction mixture is filtered and the solvent is removed in vacuo. The resulting 1-[(2'-carboxy-1'-1'-difluoro)-ethyl] IST-BOC-1SF-Tos-argininone is obtained as an oily residue, and it is then purified on HPLC according to the conditions described in Example 4.

The free carboxylic acid is converted to the symmetrical anhydride and reacted with resin-bound hexadecapeptide as described in Example 21. The two N-terminal residues of Hirulog-25, BOC-Pro and BOC-(D-Phe), are added under standard peptide synthesis conditions, and the resulting peptide is then cleaved with HF.

Example 25

Synthesis of Hiruloq- 26

Hirulog-26 has the formula: H-(D-Phe)-Pro-Argoxopropionyl-glycyl-(Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH . The hexadecapeptide, (Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu, was synthesized as described previously and allowed to remain bound to the resin. The next residue, Na<->BOC-Argoxopropionylglycine, is synthesized by reaction

the form pictured and described below.

3-(Cbz-amino)-2-oxo-3-{3-T(Ng-Tos)guanidinyl propyl}-di-tertbutylmalonate

We prepared a batch of N^-Cbz-N^Tos-arginine diazomethyl ketone in the same manner as in the preparation of JT-BOC-lSP-Tos-arginine diazomethyl ketone described in Example 21, with the exception of the substitution of N^-Cbz-lSP- Tos-arginine for N°-BOC-N<3->Tos-arginine. We dissolved 4.5 mg of N°-Cbz-Ng<->Tos-arginine diazomethyl ketone in 200 ml of CH 2 Cl 2 in a flask and cooled the solution to -70°C in a dry ice/acetone bath with stirring. Anhydrous HBr gas was then bubbled through the solution at a moderate flow rate for 15 minutes. The solution was stirred for a further 15 minutes at -70°C and then concentrated in vacuo. The resulting product, Na<->Cbz-N<9->Tos-Arg-COCH 2 Br, was recovered as 5.0 g of yellow crystals.

Meanwhile, a suspension of sodium hydride (36 mg; 80% dispersion in oil) in 1 mL of DMF and 1.2 mL of hexamethylphosphoramide ("HMPA") was added to a solution of 259 mg of di-tert-butoxymalonate in 4 mL DMF. The mixture was stirred at room temperature for 40 minutes and was then added dropwise, over 20 minutes, to a solution of 1 mmol N^Cbz-^-Tos-Arg-COCT^Br in 1 mL DNF/0.13 mL HMPA. The reaction was allowed to continue for 3 hours, and after this time the solution was poured into 50 ml of water and extracted with 2 x 50 ml of ethyl acetate. The organic phase was isolated, dried and concentrated in vacuo to an oily residue. The residue was then purified on a 3 x 10 cm silica gel column which was eluted successively with 400 ml of 5% acetone in chloroform, 400 ml of 10% acetone in chloroform and 200 ml of 20% acetone in chloroform. The fractions (25 ml) were collected and examined by TLC. Fractions containing the desired product were pooled and concentrated, yielding 3-(Cbz-amino)-2-oxo-3- {3-[(N"-Tos)guanidinyl]propyl}-di-tert- butyl malonate.

5-(Na-Cbz-amino)-4-oxo-5- {3- [ (N3-Tos) guanidinyl] propyl}

pentanoylglycine benzyl ester

The above-mentioned di-tert-butyl ester is stirred in 1.2 equivalents of IN HCl for 2 hours at room temperature. It is decarboxylated in an excess of pyridine at 100°C for 15 minutes. The solvent is then removed in vacuo, and the residue is purified by silica gel chromatography as described above. The resulting carboxylic acid is acylated with glycine benzyl ester according to the method described in example 21.

5-(amino)-4-oxo-5-{3-[ (NS-Tos)guanidinyl]propyl}pentanoylglycine

The resulting ester is dissolved in 500 ml of methanol and hydrogenated overnight at 1 atm of hydrogen gas over 600 ml of 10% palladium-carbon catalyst. The reaction mixture is filtered through celite and concentrated in vacuo to a solid residue (155 mg). The resulting amino acid is then purified by HPLC, using the conditions described in Example 4.

5-(Na-BOC-amino)-4-oxo-5-{3-[KF-Tos)

guanidinyl]propyl}pentanoylglycine

The above amino acid is converted to its corresponding BOC derivative by dissolving it in dioxane/water (2:1, v/v) and cooling to 0°C with stirring. The pH value is adjusted to 10 with 0.1 N NaOH, and then 1.1 equivalent of di-tert-butyl dicarbonate (in dioxane) is added. The mixture is stirred at 0°C to 20°C for 4 hours and then evaporated in vacuo. The residue is distributed between ethyl acetate/1% citric acid (2:1). The organic phase is isolated, extracted once with 1% citric acid and then three times with saturated NaCl. The organic phase is dried over magnesium sulfate, filtered and concentrated in vacuo to give the BOC-protected product.

The resulting protected pseudopeptide-free carboxylate is then coupled to the resin-bound hexadecapeptide using standard peptide synthesis techniques. This is followed by the sequential addition of BOC-D-Phe- and BOC-Pro to the resin-bound peptide. The complete Hirulog-26 is then cleaved from the resin, deblocked and purified as described in Example 4.

Example 26

Synthesis of Hirulog- 27

Hirulog-27 has the formula: H-(D-Phe)-Pro-Arg-(CO-CH2)-Pro-(Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu -Tyr-Leu-OH.

The (Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu hexadecapeptide was synthesized as previously described and allowed to remain bound to the resin. The remaining part of the molecule was synthesized by the reaction scheme depicted and described below.

N"-Cbz-Ng-Tos-arginine (COCH2) proline benzyl ester

We dissolved 720 mg of proline benzyl ester (HCl salt) in 25 ml of THF. This solution was then cooled to -78°C in an acetone/

dry ice bath with stirring under argon. We then added lithium diisopropylamide (8.0 mL of a 0.75 M hexane suspension) and stirred for an additional 5 minutes. To this we added 1.08 g of N^Cbz-ISF-Tos-arginine bromomethyl ketone in 10 ml of THF,

prepared as described in example 25, drop by drop over the course of 20 minutes. The reaction was stirred for a further 5 minutes, and the solution was then allowed to reach room temperature with stirring. We quenched the reaction by adding 10 ml of saturated NaCl, allowed the phases to separate and isolated the organic phase. This phase was then dried over magnesium sulphate, filtered and evaporated in vacuo.

IT-BOC-IP-Tos-arginine (C0CH2) proline

The above benzyl ester (1.3 g) was hydrogenated using the palladium carbon procedure described in Example 25. The resulting pseudodipeptide was BOC protected by the procedure described in Example 25 to give

the desired product.

The purified, protected pseudodipeptide was then coupled with the resin-bound hexadecapeptide by standard peptide synthesis techniques. Hirulog-27 was unblocked, cleaved from the resin and purified by techniques described in Example 4.

Example 27

Synthesis of Hirulocr- 2 8

Hirulog-28 has the formula: H-(D-Phe)-Pro-Arg-(CH2N)-Pro-(Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr -Leu-OH.

The (Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-0-PAM hexadecapeptide was synthesized as previously described and allowed to remain bound to the resin. The remaining part of the molecule was synthesized by the reaction scheme depicted and described below.

ISF-BOC-ISF-Tos-arginine [psiCH2N] ) proline benzyl ester

1 g of crushed 3 Å molecular sieve (Aldrich) was added to a stirred solution of 5.25 g of proline benzyl ester free base (Schweizerhall, Inc.) in 10 ml of anhydrous THF and 2 ml of anhydrous ethanol under argon at room temperature. We added 1.45 mL of 5 N methanolic HCl and 1.5 g of N^BOC-ISF-Tos-argininal (prepared as described in Example 22) to this mixture and stirred for 1 hour. An 85 mg portion of sodium cyanoborohydride was added to the mixture, and then one hour later another 85 mg portion of sodium cyanoborohydride was added. The reaction was stirred for 20 hours and filtered. We added 1 mL of water and 0.9 mL of 1 N HCl to the filtrate with stirring and then concentrated the solution in vacuo to give 6.2 g

Na-BO C-N9-Arg [psiCH-,N]-Pro-benzyl ester as a clear oil.

The oil was further purified by flash chromatography over a 5 cm flash column containing 350 ml silica gel (Merck Grade 60, 230-400 mesh, 60 Å). The product was obtained by successive elution with 0.25%, 0.75% and 1.5% methanol in chloroform.

N^-BOC-N^Tos-arginine (psiCH2N) proline

The resulting benzyl ester is hydrogenated over palladium-carbon and purified, as described in Example 25. This process gave 160 mg of N^-BOC-ISF-Arg [p_siCH2N]-proline free acid, and it was further purified using the HPLC chroma -tographic system described in Example 4, with the exception that the elution was achieved with an isocratic 26% Buffer B/74% Buffer A system, previously described in Example 22. The final yield of dipeptide was 86 mg.

The dipeptide is then coupled to the resin-bound hexadecapeptide, followed by a cycle of BOC-Pro incorporation and a cycle of BOC-(D-Phe) incorporation. Unblocking, cleavage and purification of the fully synthesized Hirulog-28 is achieved by the method described in Example 4.

Example 28

Synthesis of Hirulog- 29

Hirulog-29 has the formula: 4-chloro-isocoumarino-3-carboxy-ethoxy-(Gly) 5-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH. The (Gly) 5-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu hexadecapeptide was synthesized as previously described and allowed to remain bound to the resin. The 4-chloro-isocou-marino-3-carboxyalkyl moiety was synthesized by the reaction scheme and methods described below.

Etvl- 2- bromo- homophthalate

We mixed homophthalic acid (10.0 g), 2-bromoethanol (21.0 g) and benzene (200 ml). We then added 12-15 drops of sulfuric acid and heated under reflux for 2.5 hours. The solution was then filtered and concentrated in vacuo. The residue was washed with 250 ml of ether/hexane (1:1) and filtered on a sintered glass funnel. The resulting light brown solid was vacuum dried and approx. 15.0 g of the product.

4- Chloro- 3- r2- bromoethloxy]- isocoumarin

We mixed the ethyl 2-bromohomophthalate prepared as described above (4 g) together with phosphorus pentachloride (8.2 g) and benzene (100 ml). The mixture was heated under reflux for 4.5 hours, filtered hot and evaporated in vacuo. The red-brown oily residue was immediately chromatographed on a 24 mm x 175 mm silica gel column with dichloromethane as eluant

medium. Fractions of 20 ml were collected and examined by TLC. The 4-chloro-3-[2-bromomethyloxy]-isocoumarin eluted in fractions 2-6. The fractions were combined, evaporated in vacuo, and the resulting residue recovered as a clear pale yellow oil (2.2 g).

4- Chloro- 3- r2- oxypropanoic acid]- isocoumarin

The 4-Chloro-3-[2-bromomethyl]-isocoumarin (1.4 g) prepared above was dissolved in anhydrous THF and added directly to a refluxing solution of magnesium shavings (170 mg) and a few crystals of iodine in 15 mL of anhydrous THF, which was stirred under argon. The mixture was heated under reflux for 1.5 hours. It was then poured into an excess of dry ice in a 400 ml beaker. We let the mixture stand at 20°C until the excess of C02 had sublimed and then added approx. 100 mL of both diethyl ether and THF to the mixture, which produced a yellow solution containing a large amount of a white, coarse fine.

We bubbled anhydrous HCl through this mixture at 20°C, which dissolved most of the fines. The solution was then filtered and evaporated in vacuo to obtain the crude product. This was then recrystallized overnight from DCM.

The resulting 4-chloro-3-[3-oxypropionic acid]-isocoumarin was coupled to a glycine benzyl ester, and the resulting product was catalytically hydrogenated over palladium carbon, as described in Example 25. This pseudodipeptide was then coupled to the resin-bound hexadecapeptide, (Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu by standard peptide synthesis techniques.

Example 29

Synthesis of Hirulog- 3 0

Hirulog-30 has the formula: 4-chloro-3-[2-aminoethanol]-isocoumarin-(Gly) 5-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu. The hexadecapeptide, (Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu, is synthesized as previously described and is allowed to remain bound to the resin.

The 4-chloro-3-[2-aminoethanol]-isocoumarin moiety is prepared by a method analogous to that described in

Example 28 to synthesize 4-chloro-3-[2-bromoethanol]-isocoumarin, except that 2-aminoethanol is used instead of 2-bromoethanol in the initial step of esterifying the homof t a1 acid.

The urea bond is formed by reacting the amino group in 4-chloro-3-[2-aminoethanol]-isocoumarin with the activator, carbonyldiimidazole ("CDI"). The resulting intermediate imidazolide is not isolated, but is reacted with the resin-bound hexdecapeptide to produce Hirulog-30. Hirulog-30 is then unblocked, cleaved from the resin and purified by the techniques described in Example 4.

Example 3 0

Synthesis of Hirulog- 31

Hirulog-31 has the formula: argipidyl-(Gly) S-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH. We synthesized the hexadecapeptide, (Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu, by standard peptide synthesis techniques as previously described and left the peptide bound to the resin. The argi-pidylglycine part of this Hirulog is synthesized by the reaction scheme drawn and described below.

A dehydro-ISF-nitroargipidine is synthesized essentially by the method for synthesizing argipidine, which is described in US Patent No. 4,258,192. The only differences are that the guanidinium group is protected with a nitro function, and the heterocyclic ring in the quinoline remains unsaturated. This intermediate is used to acylate t-butylglycine by the method described in example 21. The t-butyl ester is removed by standard acid hydrolysis techniques. The resulting free acid is reacted with the hexadecapeptide using standard coupling techniques. The resulting peptide is unblocked, cleaved from the resin and purified by techniques described in example 4.

The peptide is then subjected to hydrogenation procedures described in the aforementioned US patent 4,258,192 and purified by the HPLC technique described in example 4.

Example 31

Synthesis of Hirulog- 32

Hirulog-32 has the formula: H-(D-Phe)-Pro-Arg-(Gly) 5-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH. Hirulog-32 was synthesized, purified and characterized using methods described in Example 4, except that BOC-glycine was used instead of BOC-proline in the cycle after the two cycles of BOC-glycylglycine addition.

Example 32

Synthesis of Hitulog- 33

Hirulog-33 has the formula: N-acetyl-Gly-Asp-Phe-Leu-Ala-Glu-(Gly) 3-Val-Arg-Pro-(Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu- Ile-Pro-Glu-Glu-Tyr-Leu-OH. Hirulog-33 was synthesized, purified and characterized by standard peptide synthesis techniques used in Example 4, with appropriate BOC amino acid substitutions. The CSDM portion of Hirulog-33 has an amino acid sequence identical to a segment of the fibrinopeptide A sequence of the Aa chain of human fibrinogen.

Example 33

Cleavage of Different Hirulogs by Thrombin The inhibition of thrombin by Hirulog-8 was found to be transient due to slow cleavage of the Arg-Pro bond by thrombin. After this cleavage, it was observed that thrombin regained full hydrolytic activity towards a chromogenic substrate. Therefore, Hirulog-8 was characterized as a "slow substrate" inhibitor of thrombin.

The cleavage of Hirulog, as well as other Hirulogs according to this invention, by human α-thrombin was shown in

in vitro studies. Reaction mixtures containing human α-thrombin (1.6 nM) and varying concentrations of either Hirulog-8, Hirulog-10, Hirulog-18a, Hirulog-18b,

Hirulog-18c, Hirulog-19, Hirulog-32, or Hirulog-33 (80 to 160 nM) were prepared in 20 mM Tris-HCl, pH 7.4 containing 0.1 M NaCl. Aliquots (0.975 ml) of the reaction mixtures were removed at various times and mixed in a cuvette with 0.025 ml of Spectrozyme TH (11 µM final concentration), a chromogenic substrate. The onset rate of the reaction was determined, and based on control mixtures which

contained thrombin in the absence of Hirulog, the percentage inhibition was calculated.

An alternative method used reverse-phase HPLC separation of aliquots from a Hirulog/thrombin reaction mixture. In this study, we added human α-thrombin (0.25 µM final concentration) to a reaction vessel containing one of the above Hirulogers (12.5 µM final concentration). Aliquots (50 µl) were withdrawn both before and at various times after the addition of thrombin. The aliquots were either flash-frozen or injected directly onto the HPLC column. The HPLC system used an Applied Biosystems Liquid Chromatography System equipped with an Aquapore C8 column

(0.46 x 10 cm) . The column was equilibrated in 70% solvent A (0.1% TFA in water) and 30% solvent B (0.085% TFA/70% acetonitrile and developed with a linear gradient of from 30 to 50% solvent B in 30 minutes at a flow rate of 1 ml/min. The exit stream was monitored at 214 nm. Peptide concentrations were determined by measuring the height of the sample peaks.

Both of the above investigations make it possible to determine the rate of Hirulog hydrolysis by thrombin (expressed in

m/min) and the transition rate (kcat; expressed in min"<1>). Both methods produce comparable kcat values, as shown in the table below.

As shown above, Hirulog-8, Hirulog-10, Hirulog-32 and Hirulog-3 were cleaved by thrombin with kcat values ranging from 0.056 min"<1> (slow cleavage) to 53 5 min"<1> (fast cleavage). In contrast, Hirulog-18a, Hirulog-18b, Hirulog-18c and Hirulog-19 appear to be resistant to thrombin cleavage.

Figure 5, areas A and B, shows a more detailed analysis of the cleavage of Hirulog-8 by thrombin. As depicted in Figure 5, area A, concentrations of Hirulog-8 in slight excess over thrombin exerted a transient inhibitory activity (greater than or equal to 10 minutes, depending on the Hirulog concentration). Progressively higher concentrations of Hirulog-8 showed prolonged inhibitory effects. A linear relationship between the duration of the inhibition and the Hirulog-8 concentration is shown in Figure 5, area B. From these data, we calculated the transition time, or coat, to be 0.37 min"<1>: By purification and sequence analysis of the Hirulog- 8-derived reaction products that appeared in the reactions above, we demonstrated that Hirulog-8 was slowly cleaved by thrombin

at the Arg-Pro bond. This is a highly unusual cleavage site for serine proteases, and we believe that it is susceptible to cleavage in Hirulog-8 due to the peptide's high affinity for thrombin.

Example 34

The effect of the coupler length

on the activity of Hirulog

Hirulog-8, Hirulog-13, Hirulog-15 and Hirulog-16 differ from each other only by the length of the polyglycine moiety in their respective linker segments. To determine the effect of linker length on activity, we compared the inhibition of human α-thrombin on each of these Hirulogs. The following table indicates the connector length on each of these Hirulogs.

The antithrombin activities of these Hirulogs were measured regarding thrombin-catalyzed hydrolysis of Spectrozym TH mainly as described in example 9. Figure 6 depicts the relationship between linker length and Kx for Hirulog inhibition of this thrombin-catalyzed reaction. This figure shows that Hirulog-8, Hirulog-15 and Hirulog-16 have comparable inhibitory activities, while Hirulog-13, with an 18Å linker length, has an activity reduced to less than 1/10. This confirms that linker lengths of >18Å and <42Å do not affect Hirulog activity. Although the applicants do not wish to be bound by theory, they believe this is due to the fact that the Hirulog linker is as disordered when free in solution as it is when bound to thrombin. The applicants also believe that there is little cooperation between the CSDM and ABEAM parts of the thrombin inhibitors in binding to thrombin according to this invention.

Example 35

Inhibition of thrombin catalyzed

hydrolysis by various Hirulogs

We compared the inhibitory activity of different thrombin inhibitors prepared according to the present invention on thrombin-catalyzed hydrolysis of a tripeptidyl-p-nitro-anilide substrate. The antithrombin activities of Hirulog-10, Hirulog-18a, Hirulog-18b, Hirulog-18c, Hirulog-19, Hirulog-32 and Hirulog-33 were investigated by the method described in Example 9, using Spectrozyme TH as substrate. The table below shows the calculated K: values as well as the Pj^-Pj^' sequence for each of these thrombin inhibitors.

As indicated above, Hirulog-10 and Hirulog-32 were poor inhibitors of thrombin-catalyzed hydrolysis of Spectrozyme TH. This was consistent with the fact that each of these inhibitors was rapidly cleaved by thrombin at the Pj;-Pi' bond. In Hirulog-19, where this bond was reduced to the psiCH?-NH linkage and rendered non-cleavable by thrombin, effective inhibition of thrombin hydrolysis was observed.

The studies with £-homoarginine-containing inhibitors (Hirulogene 18a, -18b and -18c) showed that this amino acid derivative can replace arginine in the inhibitors produced according to this invention without influencing the activity. Furthermore, this shows that replacement of the amide bond with a methylene group does not reduce thrombin's inhibitory activity to a particular extent. The 30- to 50-fold increase in Kx for Hirulog-18c, compared to Hirulog-18a and Hirulog-18b, respectively, suggests that the structure of the P'i amino acid is important in inhibitory activity. Without wishing to be bound by theory, Applicants believe that the presence of the phi-psi angles in the P'<1->amino acid (Gly in Hirulog-18a; Pro in Hirulog-18b) as well as structural bonding (caused by the proline in Hirulog -18b) contributes to the potency of the inhibitors produced according to this invention. An alternative possibility is that the S-branched side chain in the P'<1-> amino acid Val in Hirulog-18c may weaken the binding of the CSDM portion of that molecule to the thrombin-reactive center due to steric considerations.

Example 36

Binding of Hirulog-8 to

the active site in thrombin Diisopropylfluorophosphate (DFP) is a well-known inhibitor of serine proteases, including thrombin, which works by covalently modifying the hexyl group at Ser-195. We added a 270-fold excess of <14>C-DFP to thrombin, in 0.1 M sodium borate, pH 8.0. After 10 minutes of reaction, formation of a thrombin complex was shown by SDS-PAGE and fluorographic analyzes (Figure 7, lane 1). When the reaction was carried out in the presence of Sulfo-Tyr63-N-acetyl-hirudin53.64 (at 300- and 3000-fold molar excess over thrombin), the modification of thrombin by [14C]-DFP was not significantly changed (Figure 7, lanes 4 and 5). However, when we performed the reaction in the presence of Hirulog-8 (at a 3- or 30-fold molar excess over thrombin), the incorporation of [14C]-DFP into the thrombin-catalyzed site was completely blocked (Figure 7, lanes 2 and 3). . These data show that CSDM in the thrombin inhibitors produced

according to this invention is able to bind to the catalytic site in thrombin and inhibit catalytic activity.

Example 3 7

Comparison of antithrombin activity

of Hirulog-8 and a synthetic catalytic site-directed pentapeptide (D-Phe-Pro-Arg-Pro-Gly

As shown in Figure 1, Hirulog-8, in contrast to its constituent anion-binding outward site-associating unit Sulfo-Tyr63-N-acetyl-hirudin53_64, was able to inhibit throbin-catalyzed hydrolysis of small p_-nitroanilide substrates. In a similar way, we have investigated the ability of a (D-Phe)-Pro-Arg-Pro-Gly pentapeptide to inhibit thrombin catalytic reactivity.

The pentapeptide was synthesized as described in Example 4 using a BOC-lysine-divinylbenzene resin. The pentapeptide was purified and characterized as described in Example 4.

The effects of both Hirulog-8 and this pentapeptide on thrombin-catalyzed hydrolysis of Spectrozyme TH were examined as described in Example 9, using fixed peptide concentrations of 50 nM or 10 µM, respectively. Our results show that while nanomolar concentrations of Hirulog-8 can inhibit the thrombin-catalyzed reaction, concentrations of pentapeptide as high as 10 µM have no significant effect on the thrombin-catalyzed rate. These data show that the CSDM component in the thrombin inhibitors produced according to the present invention is in itself only a weak inhibitor of thrombin's catalytic function.

Example 3 8

In vivo anticoagulant activity of Hiruloq-8

We determined the in vivo anticoagulant activity of Hirulog-8 after intravenous administration of this peptide to baboons. We used different dosages of Hirulog-8 varying from 0.002 to 0.2 mg/kg/min. Baboons (male, 10-15 kg) were sedated with ketamine hydrochloride prior to administration of Hirulog-8. Whole blood from treated and control animals was removed from a catheter placed in the femoral vein and collected in 3.8% sodium citrate (9:1; blood:sodium citrate). Plasma was obtained by standard methods, and APTT was recorded by methods described in Example 10. As shown in Figure 8, Hirulog-8 produced a dose-dependent increase in the APTT value. A 200% increase in the APTT value (considered therapeutic range) was achieved with the lowest Hirulog dose (infusion 0.002 mg/kg/min).

Example 39

Inhibition of coagulate-bound thrombin by Hirulog-8

It is known that thrombin can bind to a fibrin clot, and once absorbed, it continues to cleave additional fibrinogen, resulting in growth of the clot. Coagulated thrombin has been shown to be resistant to neutralization by the heparin-antithrombin II complex [P. J. Hogg et al., "Fibrin Monomer Protects Thrombin From Inactivation By Heparin-Antithrombin III: Implications for Heparin Efficacy", Proe. Nati. Acad. Pollock. USA, 86, pp. 3619-23 (1989)], but it can be inhibited by antithrombin III-independent inhibitors, such as PPACK, hirudin or Sulf o-Tyr63-N-acetyl-hirudin53,64. Clot-bound thrombin is thought to play a role in thrombus growth and in the recovery of thrombosis after thrombolytic therapy.

We compared Hirulog-8's and heparin's ability to inhibit clot-bound thrombin and used the method described by J. I. Weitz et al., "Clot-Bound Thrombin Is Protected from Heparin Inhibition -- A Potential Mechanism for Rethrom-bosis After Lytic Therapy", Blood, 74, p. 136a, (1989).

A clinically relevant dose of heparin (0.1 U/ml) inhibited fibrinopeptide A (FPA) release catalyzed by soluble thrombin by approximately 70%. However, a similar dose had no effect on the FPA release catalyzed by clot-bound thrombin. In contrast, Hirulog-8 had an almost identical inhibitory effect on FPA release catalyzed by either soluble or clot-bound thrombin (Figure 9).

This study showed that Hirulog-8, as well as the other thrombin inhibitors of this invention, are more effective than current drugs in blocking thrombus aggregation, in increasing the rate of thrombolytic reperfusion, and in preventing the recurrence of thrombi after thrombolytic treatment.

Example 40

The effect of Hirulog-8 on in vivo platelet-dependent thrombosis

Because baboons are known to have similar coagulation and hemostatic responses to humans, we used a baboon model to determine the ability of Hirulog-8 to interrupt platelet-dependent thrombosis. Specifically, we placed different thrombogenic surfaces into a permanent external AV bypass conduit in the animals. These surfaces included segments of end-arterectomized baboon aorta, collagen-coated silastic tubing material, collagen-coated vascular grafts of "Gortex" and "Dacron". After placement of the bypass line, the surfaces were exposed to naturally flowing blood to induce thrombus formation. We measured the formation of thrombi over a period of 60 minutes by monitoring the shedding of platelets on the thrombogenic surface. These measurements were recorded by external gamma camera imaging after pre-infusion of the experimental animal with autologous 1:L1In-labeled platelets.

Placement of a 5 cm segment of end-arterectomized baboon aorta into the externally placed AV bypass in the absence of Hirulog-8 led to a time-dependent deposition of platelets. This collection reached a plateau within 60 minutes, at which time a total of 14.0 + 5.0 x 10<8> platelets were found deposited on the end-arteriectomized segment. Doses of 0.002 and 0.01 mg/kg/min of Hirulog-8 inhibited platelet excretion by 53.6% and 75.5%, respectively. These results are depicted in Figure 10. The ED50 value for Hirulog-8 (the dose required to reduce platelet shedding by 50%) in this system was 0.002 mg/kg/min.

When we placed 5 cm segments of collagen-coated silastic tubing material into the AV bypass, 12.6 + 5.0 x IO<8 >platelets/cm were deposited after 60 minutes in the absence of Hirulog-8. Administration of Hirulog-8 resulted in a dose-dependent inhibition of platelet deposition. A dosage of 0.04 mg/kg/min of Hirulog-8 completely inhibited platelet deposition. The results of this part of the experiment are shown in Figure 11. The ED50 value for Hirulog-8 in this system was calculated to be 0.004 mg/kg/min.

Both collagen-coated vascular grafts of "Gortex" or "Dacron" are known to be more thrombogenic than silastic tubing material. A total amount of 3 5.0 + 6.0 x 10<8> was deposited on the "Gortex" tube after a 60 minute exposure to native blood in the absence of Hirulog-8. We found that Hirulog-8 again showed a dose-dependent antithrombotic effect against the formation of platelet thrombi. A dose of 0.2 mg/kg/min Hirulog-8 caused 62.9% inhibition of platelet aggregation. The ED50 value for Hirulog-8 in the Gortex system was 0.135 mg/kg/min. A similar result was obtained for Dacron grafts. The higher dosage of Hirulog-8 required to inhibit platelet shedding on these two surfaces was to be expected due to their high thrombogenic activity.

We also determined the effect of Hirulog-8 on platelet-dependent thrombus formation at both high and low shear force, using a two-chamber device that enabled simultaneous measurements of both shear force conditions. The device was composed of a 2 cm segment of collagen-coated "Cortex", followed by 2 cm segments of expanded diameter. With this device, thrombus formation was initiated by exposure to naturally flowing blood on a segment of collagen-coated Gortex tubing at high shear. This part of the experimental procedure simulated artery-like conditions. As the blood entered the enlarged diameter segments, low shear vortex conditions were maintained, thereby simulating venous thrombosis. In control animals, a total of 9.3 + 2.3 x IO<8> and

6.1 + 0.5 x IO<8> platelets/cm at 40 minutes in the high and low shear segments. Hirulog-8 inhibited platelet deposition in both high- and low-shear segments in a dose-dependent manner. A dose of 0.5 mg/kg/min inhibited platelet aggregation by 42.6% at low shear and by 29.0% at high shear.

Example 41

Comparison of Hirulog-8 with other antithrombotic agents in inhibiting acute platelet-dependent thrombosis

We examined the effect of heparin, low molecular weight heparin, and recombinant hirudin on platelet deposition in the collagen-coated silastic conduit/external AV bypass conduit in the baboon model described in Example 40.

It has previously been shown that heparin administered as a 160 U/kg bolus injection followed by a 160 U/kg/h infusion inhibited platelet deposition to a level of approx. 80% of what was observed in a saline-treated control animal. Low-molecular-weight heparin, given as a bolus injection of 53 anti-Xa U/kg, followed by infusion at 53 anti-Xa U/kg/h, gave similar results. [Y. Cadroy, "In Vivo Mechanism of Thrombus Formation. Studies Using a Primate Model", Doctoral Thesis, L'Universite Paul Sabatier de Toulouse

(Sciences)

(1989)]. At equivalent molar doses (5 nmol/kg/min), recombinant hirudin [A. B. Kelley et al., "Recombinant Hirudin Interruption of Platelet-Dependent Thrombus Formation", Circulation, 78, pp. 11-311 (1989)] both inhibited platelet-dependent thrombus formation by 60-70% compared to the control. These results are depicted in Figure 12. Other thrombin inhibitors have previously been investigated in the baboon model [A. B. Kelley et al., "Comparison of Antithrombotic and Antihemostatic Effects Produced by Antithrombins in Primate Models of Arterial Thrombosis", Thromb. and Hemostas., 62, p. 42 (1989)]. The reported ED50 doses of these agents on collagen-coated surfaces, as well as our ED50 determinations, are summarized in the table below.

Example 42

The effect of Hirulog-8 on fibrin secretion

We measured the effect of Hirulog-8 on the deposition of fibrinogen) in the thrombi formed in the end-arterectomerized aortic and collagen-coated silastic tube segment model system described in Example 40. The fibrin deposition was determined by measuring 125I-fibrin ( ) 30 days after the i:L1In platelet assay described above was completed. This allowed the 111 - In isotope to decay to a non-disturbing level.

Figure 13 shows that in the absence of Hirulog-8, 0.17 mg/cm fibrin was deposited on the collagen-coated tube material after 60 minutes of exposure to liquid blood described in Example 40. Doses of 0.01 and 0.04 mg/kg/min inhibited complete fibrin(ogen) deposition. Similar results were obtained with the end-arterectomized aortic segment model. These results show that the thrombin inhibitors prepared according to this invention are effective in reducing fibrin(ogen) deposits associated with a thrombus, as well as in blocking acute platelet-dependent thrombus formation.

Example 43

Measurement of the clearance time for Hirulog-8

We used a baboon model to determine the clearance times of Hirulog-8 following intravenous infusion, single intravenous bolus injection and single subcutaneous bolus injection. APTT assays, performed as described in Example 11, were used to monitor clearance times.

We administered different dosages of Hirulog-8 (0.002-0.2 mg/kg/min) to baboons via systemic intravenous infusion, over a period of 60 minutes. APTT was measured after the 60-minute infusion and at various time intervals thereafter. We calculated that the average half-life for Hirulog-8 clearance was 9.2 + 3.3 minutes.

To determine the clearance time after a single bolus injection, we injected baboons with a dose of 1 mg/kg Hirulog-8 intravenously or subcutaneously. APTT measurements were made at different time intervals after the injection. Figure 14 shows that APTT increased to a peak of 570% of the control value 2 minutes after intravenous injection. The half-life of Hirulog-8 after intravenous injection was 14 minutes. Figure 15 shows that at the earliest time point after subcutaneous injection of Hirulog-8 (i.e. 15 minutes) the APTT had increased to approx. 200% of the control. Clearance by the subcutaneous route was prolonged to a half-life of 340 minutes. Hirulog-8 administered subcutaneously was found to be quantitatively adsorbed.

Example 44

Effect of Hirulog-8 in baboon models

on disseminated intravascular coagulation

We induced septicemia in baboons by injection of a lethal dose of live E. coli according to the procedure described by F. B. Taylor et al., J. Clin. Invest., 79, pp. 918-25 (1987). Hirulog-8 was infused at a dose of 0.08 mg/kg/h starting 15 minutes before the injection of EL. coli until up to 6 hours after the injection. In the absence of Hirulog-8, Ej led. coli-induced septic shock to a noticeable decrease in the neutrophil count, blood pressure and hematocrit value. Control animals showed a decrease in hematocrit to 70% of baseline and a drop in blood pressure to 20% of baseline after 3 hours. Administration of Hirulog-8 completely attenuated the drop in hematocrit and limited the drop in blood pressure to 60% of baseline.

Despite attenuation of DIC by Hirulog-8, the lethal infusion of E_j_ coli still resulted in death. An autopsy of both the control and the Hirulog-8 treated animals revealed massive tissue edema in both groups, but only the control group showed intravascular thrombosis. The results of the autopsies show that the interruption of the coagulopathic stage in septicemia alone is not sufficient to prevent death due to septic shock. *

Example 45

The effect of a combination of tPA

and Hirulog-8 on thrombolysis

To determine the effect of Hirulog-8 on enhancing tPA-induced thrombolysis, we used a rat model of arterial thrombolysis. In this model, an experimental thrombus was formed in the abdominal aorta after balloon catheter denudation and severe (95%) stenosis. Blood flow and blood pressure were recorded remote from the site of the injury and stenosis.

We randomized rats to receive tPA (0.1 mg/kg bolus followed by 1.0 mg/kg/h infusion) along with one of the following: saline, heparin (10 U/kg bolus, followed by 1.5 U /kg/min infusion), recombinant hirudin (1.0 mg/kg bolus followed by 0.02 mg/kg/h infusion) or Hirulog-8 (0.6 mg/kg bolus followed by 0.02 mg/kg/ t infusion). The anti-thrombotic agent and saline were administered simultaneously with the tPA and for an additional 50 minutes after the end of the tPA infusion.

Figure 16 shows the results of these experiments. Animals treated with tPA plus saline showed reperfusion times of 16.2 minutes. Heparin reduced the reperfusion time to 12.2 minutes, while recombinant hirudin reduced it to 13.0 minutes. None of these reductions were statistically significant (p < 0.05). The combination of Hirulog-8 and tPA significantly reduced the reperfusion time to 4.4 minutes

(p < 0.01), thus accelerating the fibrinolytic action of tPA by a factor of four.

Heparin, hirudin, and Hirulog-8 all significantly prevented reocclusion, compared to saline-treated controls (Figure 17). Each of these agents also prolonged APTT to values of 600%, 500% and 400% respectively in relation to the control values (figure 18). Moreover, both heparin, hirudin and Hirulog-8 increased the time for vessel patency to values of 80.2%, 82% and 93.1% respectively (control = 43.6%) (figure 19). These results show that the thrombin inhibitors produced according to the present invention are superior to known antithrombotics when it comes to increasing the effect of tPA.

Example 46

Effect of Hirulog-8 and other antithrombotic agents on bleeding time in baboons

We used standard bleeding time measurement to investigate the effects of Hirulog-8 on hemostasis.

Different dosages of Hirulog-8 (0.002 to 0.2 mg/kg/min) were analyzed for their effect on bleeding time. Doses of 0.002 to 0.04 mg/kg/min caused no significant increase in bleeding times. The results of this experiment are depicted in Figure 20. At a dose of 0.1 mg/kg/min, Hirulog-8 causes a two-fold increase in bleeding time compared to control values. At 0.2 mg/kg/min Hirulog-8, the bleeding times increased to 3 times the control values. These results clearly show that dosages necessary to inhibit platelet-dependent thrombosis (0.002 mg/kg/min; see Example 40) do not cause a significant effect on hematostatic clot formation.

We also examined the effects of a selection of other agents on the standard bleeding time in baboons, as well as on the systemic anticoagulant effects (measured by APTT). These results are summarized below:

Example 47

Synthesis of Hirulog- 34

Hirulog 34 has the formula: H-(D-Phe)-Pro-Arg-(tetraethylene-glycolylsuccinyl)-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH. The decapeptide Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu is synthesized as described previously and is allowed to remain bound to the resin. The remaining part of the molecule is synthesized by the reaction scheme depicted and described below:

Na<->BOC-(D-Phe)-Pro-N<9>,N9-(Cbz) 2-Arg

Na-B0C-N9-(Cbz)2-Arg (Bachem, Inc., Torrance, CA) is reacted with an excess of ethereal diazomethane and then treated with acid to remove the N"-BOC group. The resulting product, N< 9>,N<9->(Cbz)2-arginine methyl ester, then dissolve 1 DMF, cool in an ice bath and treat successively with 2 equivalents of N°-BOC-proline, 1 equivalent of butanol, 1 equivalent of EDCI and 1 equivalent diisopropylethylamine. The reaction mixture is stirred overnight, then diluted with 5 parts by volume of cold water and extracted with ethyl acetate. The organic phase is recovered and washed successively with equal parts by volume of saturated citric acid, saturated NaHCO 3 and saturated NaCl. The product is dried over magnesium sulfate and concentrated in vacuo. resulting dipeptide intermediate, N"-BOC-prolyl-N9,N9-(Cbz) 2-arginine methyl ester, is treated with a tenfold molar excess of 4N HCl/dioxane for 30 minutes. The free HCl is removed in vacuo, and the residue is dissolved in anhydrous DMF. The product is then cooled in an ice bath and reacted with two equivalents of ISP-BOC-(D-Phe) in the presence of butanol, EDCI and diisopropylethylamine, as described above. The crude, orthogonally protected tripeptide is isolated as described above and purified on a silica gel column eluted with chloroform:methanol (95:5) containing 0.1% NH 4 OH. The N<"-BOC-(D)-phenylalanylprolyl-N^I^-^bz) 2-arginine methyl ester is saponified with 2 equivalents of LiOH in methanol:water (2:1) at room temperature for 3 hours. The methanol is removed in vacuo, and the aqueous solution is washed with 2 volumes of diethyl ether. The solution is acidified to pH 3 with saturated citric acid. The resulting untreated tripeptide as free acid is extracted into ethyl lactate. The organic phase is washed with 3 volumes of saturated NaCl, dried over anhydrous magnesium sulfate and concentrated in vacuo. resulting Na<->B0C-(D)-phenylalanylprolyl-N^N<9-> (Cbz) 2-arginine is purified by reverse phase HPLC under conditions described in Example 4.

Na-BOC-(D)-phenylalanylprolyl-N^N9-(Cbz)2-

arcrinine tetraethylene glycol ester

A solution of the above tripeptide is dissolved in THF and esterified with 1.5 equivalents of tetraethylene glycol in the presence of 1 equivalent of both diethylazodicarboxylate and triphenylphosphine, as described in 0. Mitsunobu, "The Use of DiethylAzodicarboxylate and Triphenylphosphine in Synthesis and Transformation of Natural Products" , Synthesis, pp. xl-28 (1981).

Na-B0C-(D)-phenylalanylprolyl-N^N3-(Cbz)2-arginine- tetraethyleneglycol- hemisuccinate

The resulting compound is dissolved in DMF and esterified with 1 equivalent of succinic anhydride in the presence of 1 equivalent of diisopropylethylamine. The volatile solvents are removed in vacuo, and the free acid is purified by reverse-phase HPLC under conditions described in example 4.

Na-B0C-(D)-phenylalanylprolyl-N^N9-(Cbz) 2-arginine-tetraethyleneglycol-hemisuccinate-N-hydroxysuccinimide ester

A solution of the above acid is mixed with 1 equivalent of N-hydroxysuccinimide in DMF, cooled in an ice bath and mixed with 1 equivalent of DCC in DMF, added dropwise. The reaction is stirred at room temperature for 24 hours, filtered to remove the precipitated dicyclohexylurea and concentrated in vacuo. The solution is concentrated with cold benzene/hexane to give the crude peptide glycol-conjugated N-hydroxy-succinimide ester.

The above-mentioned compound is reacted with the resin-bound dodeca-peptide, as described in example 21. The resulting Hirulog-34 is cleaved from the resin, purified and characterized as described in example 4.

While in the above we have presented a number of embodiments of this invention, it is obvious that our basic embodiment can be changed to provide other embodiments. Therefore, it will be understood that the scope of this invention is to be defined by the accompanying claims and not by the specific embodiments that have been presented above by way of examples.

Claims (10)

1. Analogy method for the production of therapeutically active peptides or salts thereof, where the peptides comprise: a) a catalytic site-directed unit that binds to and inhibits the active site of thrombin; wherein said catalytic site-directed unit is selected from units of formula X-A-1-Aj-Aj-Y or X-C^-C^-Aj-Y, wherein X is hydrogen or is a chain comprising from 1 to 35 atoms; A1 is Arg, Lys or Orn; A2 is a non-amide bond; A3 is a stem comprising from 1 to 9 atoms; Y is a bond; Cx is a derivative of Arg, Lys or Orn comprising a carboxylate unit reduced or displaced from α-carbonate by a unit comprising a stem comprising from 1 to 10 atoms; and C 2 is a non-cleavable bond; b) a linker unit comprising a stem with a calculated length of between about 18Å and 42Å; and c) an anion-binding exosite-associating unit that binds to the anion-binding exosite of thrombin, wherein the anion-binding site-directing unit is selected from units of formula: where W is a bond; Bx is an anionic amino acid; B2 is any amino acid; B3 is Ile, Val, Leu, Nie or Phe; B4 is Pro, Hyp, 3,4-dehydroPro, Thiazolidine-4-carboxylate, Sar, any N-methyl amino acid or D-Ala; B5 and B6 are any anionic amino acids; B7 is a lipophilic amino acid selected from the group comprising Tyr, Trp, Phe, Leu, Nie, Ile, Val, Cha, Pro or a dipeptide comprising one of these lipophilic amino acids or and any amino acid; B8 is a bond or peptide containing from one to five residues of any amino acid; and Z is a carboxy-terminal residue selected from OH, C 1 -C 8 alkoxy, amino, mono- or di-(C 1 -C 2 ) alkyl-substituted amino or benzylamino; wherein said catalytic site-directing unit is bound to said linker unit which is bound to said anion-binding exosite-associating unit, characterized in that the method comprises the step of synthesizing said thrombin inhibitor by solid-phase synthesis, solution-phase synthesis, mixed heterologous/solid-phase synthesis or a combination thereof. 2. Process according to claim 1, where B1 is Glu, B2 is Glu, B3 is Ile, B4 is Pro, B5 is Glu, B6 is Glu, B7 is Tyr-Leu, Tyr (SO3H)-Leu, Tyr (OS03H)- Leu or (3,5-diiodoTyr)-Leu, B8 is a bond and Z is OH characterized in that corresponding starting material is used. 3.. Method according to claim 1, where the stem of said linker unit consists of any combination of atoms selected from the group comprising carbon, nitrogen, sulfur and oxygen, characterized in that corresponding starting material is used. 4. Method according to claim 3, where the linker comprises the amino acid sequence Gly-Gly-Gly-Asn-Gly-Asp-Phe, characterized in that corresponding starting material is used. 5. Method according to claim 1, where the catalytic site-directed unit comprises the formula X-A1-A2-A3-Y, characterized in that corresponding starting material is used. 6. Method according to claim 5, where X is D-Phe-Pro; A1 is Arg and A3 is D-Pro, Pro or Sar, characterized in that corresponding starting material is used. 7. Method according to claim 6, where the thrombin inhibitor is selected from the group comprising Hirulog-8 (H-(D-Phe)-Pro-Arg-Pro-(Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr -Leu-OH) and Hirulog-12 (H-(D-Phe)-Pro-Arg-Pro-(Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr (0S-03)-Leu-OH), characterized in that corresponding starting material is used. 8. Method according to claim 5, where X is N-acetyl-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val; A1 is Arg and A3 is Pro, where said thrombin inhibitor is Hirulog-33 (N-acetyl-Gly-Asp-Phe-Leu-Ala-Glu-(Gly) 3-Val-Arg-Pro (Gly) 4-Asn-Gly -Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH), characterized in that corresponding starting material is used. 9. Method according to claim 1, where the catalytic site-directed unit is encompassed by the formula characterized in that corresponding starting material is used. 10. Method according to claim 1, wherein the thrombin inhibitor is selected from the group comprising Hirulog-18a (H-(D-Phe)-Pro-(/3-homoarginine)-(Gly) 5-Asn-Gly-Asp-Phe -Glu-Glu_ile-Pro-Glu-Glu-Tyr-Leu-OH) and Hirulog-18b (H (D-Phe) -Pro-(/3-homoarginine) - (Gly) 4-Asn-Gly-Asp-Phe -Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH), characterized in that corresponding starting material is used.